Yeasts

Genomics, 1992 May, 13(1), 75 - 80
Sequence-tagged sites (STSs) spanning 4p16.3 and the Huntington disease candidate region; Gusella JF et al.; The generation of sequence-tagged sites (STSs) has been proposed as a unifying approach to correlating the disparate results generated by genetic and various physical techniques being used to map the human genome . We have developed an STS map to complement the existing physical and genetic maps of 4p16.3, the region containing the Huntington disease gene . A total of 18 STSs span over 4 Mb of 4p16.3, with an average spacing of about 250 kb . Eleven of the STSs are located within the primary candidate HD region of 2.5 Mb between D4S126 and D4S168 . The availability of STSs makes the corresponding loci accessibility to the general community without the need for distribution of cloned DNA . These STSs should also provide the means to isolate yeast artificial chromosome clones spanning the HD candidate region.

Mol Cell Biol, 1992 May, 12(5), 2295 - 301
A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin; Fleig UN et al.; The cdc2 gene product, a 34-kDa phosphoprotein with serine/threonine protein kinase activity, has been implicated as the key component in the regulation of the eucaryotic cell cycle . Activation of the cdc2 protein kinase is regulated by its phosphorylation state and by interaction with other proteins . We have mutagenized the fission yeast cdc2 gene to obtain conditionally dominant negative alleles . One of these mutants, named DL2, is characterized in this report . Overexpression of the mutant protein in a wild-type cdc2 background is lethal and leads to arrest in the G2 phase of the cell cycle . The mutant phenotype is the result of a single amino acid change in the GDSEID motif of the protein, a region of identity in all cdc2 homologs, and results in a nonfunctional protein that shows an altered content of phosphothreonine . Multicopy suppressors of the dominant negative phenotype have been isolated, and one of these has been shown to encode the cdc13 cyclin B gene product.

Biol Mass Spectrom, 1992 May, 21(5), 242 - 8
Biosynthesis and characterization of 3-hydroxyalkan-2-ones and 2,3-alkanediols: potential products of aldehyde metabolism; Montgomery JA et al.; A mass spectrometric study of an enzymatic synthesis of 3-hydroxyalkan-2-ones (acyloins) is presented . Incubation of pyruvate or (13C3)pyruvate and various alkanals in the presence of pig heart pyruvate dehydrogenase or yeast pyruvate decarboxylase resulted in the formation of acyloins with chains two carbons longer than the alkanals . Product formation was rapid for all saturated aldehydes with chain lengths from 2 to 12 carbons . Incubation with 2,3-unsaturated aldehydes did not produce condensation products . Reduction of acyloins with sodium borohydride produced the corresponding 2,3-alkanediols . Analysis by gas chromatography/mass spectrometry was used to characterize the 3-hydroxyalkan-2-ones as the oxime-trimethylsilyl derivatives and the 2,3-alkanediols as the bistrimethylsilyl derivatives.

New Biol, 1992 May, 4(5), 512 - 9
Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein; Gegonne A et al.; The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli . We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1 . The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in all known members of the Ets family . Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein . This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4 . In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family . These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators.

Ann Hum Genet, 1992 May, 56 ( Pt 2), 93 - 7
Carrier detection of deletions of the Hunter gene by in situ hybridization; Stone S et al.; Deficiency of the lysosomal enzyme alpha-iduronate sulphate sulphatase (IDS) causes the clinical manifestations of Hunter syndrome, an X-linked condition . In about 30% of male patients, the disease is due to a major deletion . Using a non-isotopic in situ hybridization (NISH) method, and a yeast artificial chromosome (YAC) probe, the Hunter gene was mapped to the terminal region of the human X chromosome, close to the Xq28 band . The NISH procedure was then applied to investigate the carrier status of female relatives of a Hunter patient known to have a deletion of the IDS gene . Unequivocal evidence that two female relatives were carriers of the deletion was obtained, demonstrating that the NISH method is a valuable diagnostic tool in genetic counselling of families with Hunter patients.

J Natl Med Assoc, 1992 May, 84(5), 449 - 52
Localized pulmonary disease due to Trichosporon beigelii; Qadri SM et al.; A case of pulmonary infection caused by Trichosporon beigelii is reported . The infection occurred in a neutropenic patient with acute lymphoblastic leukemia . His chest radiograph showed a 6-cm pulmonary infiltrate in the right midzone and an apical infiltrate on the left . Repeated cultures of bronchoalveolar lavage grew budding yeast that was identified as T beigelii on the basis of morphological, cultural, and biochemical characteristics . He responded to amphotericin-B therapy . Systemic infections caused by this yeast are rare and its causal relationship in localized lung disease has been reported only seven times previously.

Srp Arh Celok Lek, 1992 May-Jun, 120(5-6), 184 - 7
{Pityriasis versicolor--modern views on etiology, pathogenesis and therapy}; Karadaglic Dj; Pityriasis versicolor (Tinea versicolor) is a superficial chronic fungal infection caused by Pityrisporum species which are normal "inhabitants" of the cutaneous flora . The morphologic changes from yeast to mycelial hypha form are important in the development of clinical lesions . The onset and course of the disease are under the influence of genetic factors, age, sex, climate, local environmental factors, malnutrition, pregnancy, oral contraceptives, corticosteroid and immunosuppressive treatment . They favorize transformation of saphrophytic to pathogenic form, and are the cause of recurrences and chronicity of the disease . The ultrastructural and immunologic studies which are carried out today would significantly contribute to a better understanding of pathogenesis of the disease . The disease is limited to seborrheic areas of the skin and commonly has three clinical forms: papulosquamous, follicular and inverse . This can make great problems in differential diagnosis . In the treatment of these patients it should be kept in mind that Pityriasis versicolor is not a primary contagious disease and that conversion of Pityrisporum depends on the predisposing factors . Numerous medicaments with local and systemic effect which are used nowaday in the treatment and prevention of pityriasis are reported . Way of application, doses, duration of therapy, advantages and disadvantages are given in detail.

New Biol, 1992 May, 4(5), 551 - 7
FLP-mediated intermolecular recombination in the cytoplasm of Drosophila embryos; Konsolaki M et al.; We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT . Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration . The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo . This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable . Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos.

EMBO J, 1992 May, 11(5), 1949 - 55
Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates; Lanzer M et al.; We have mapped Plasmodium falciparum erythrocytic stage transcription units on chromosome 10 in the vicinity of the gene encoding the glycophorin binding protein (GBP130) using yeast artificial chromosomes (YACs) . Three erythrocytic stage transcription units are clustered in a 40 kb region . Two of these genes are closely linked, separated by less than 2 kb . Nuclear run-on data demonstrate that transcription of these two genes, though unidirectional, is monocistronic . Within this intergenic region are the sites at which transcription of the upstream gene terminates and the GBP130 gene initiates . These studies represent the first description of the minimal and necessary cis-acting elements for transcription termination and initiation in this protozoan parasite.

Trends Genet, 1992 May, 8(5), 174 - 80
Ustilago maydis, the delightful blight; Banuett F; Recent studies of the corn smut fungus life cycle and its regulation by two mating type loci and other genes provide a cornucopia of challenges in cell biology, genetics and protein structure . The fungus can exist in two states: nonpathogenic and pathogenic . The change from one state to the other is accompanied by a change in morphology (yeast-like to filamentous) and growth properties (saprophytic to parasitic).

Genomics, 1992 May, 13(1), 16 - 20
A 14-Mb physical map of the region at chromosome 11q13 harboring the MEN1 locus and the tumor amplicon region; Tanigami A et al.; We have constructed a physical map of chromosome 11q13, using 54 DNA markers that had been localized to 11q13.1----q13.5 by means of somatic hybrid cell panels . Although the map has some gaps, it spans nearly 14 Mb and includes the region containing the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and also the region that is amplified in several types of malignant tumors . As the estimated average distance between each locus is roughly 300 kb, the markers reported here will be valuable resources for construction of contig maps with yeast artificial chromosomes and/or cosmid clones . Furthermore, these clones will be useful in efforts to identify the MEN1 gene and in analyses of the amplification units present at 11q13 in certain tumors.

Biochem Biophys Res Commun, 1992 Apr 30, 184(2), 986 - 92
Contribution of the p51 subunit of HIV-1 reverse transcriptase to enzyme processivity; Huang SC et al.; Human immunodeficiency virus Type I reverse transcriptase is active as either the homodimer (p66/p66) or the heterodimer (p66/p51) . Purified recombinant p66 and p51 expressed in yeast were reconstituted in the presence of 60 mM sodium pyrophosphate to enhance dimer formation . Comparison of the processivity of these two active reconstituted forms shows that the heterodimer is more processive than the homodimer with a cycle almost twice as long as judged by assays utilizing poly (U,G) as a challenger to primer-template . Binding assays demonstrated that the heterodimer has a higher affinity for primer-template than the homodimer and that the p51 subunit has an affinity equal to that of the heterodimer . These results suggest that the p51 subunit functions to increase processivity in the heterodimer.

Carbohydr Res, 1992 Apr 27, 228(2), 377 - 98
Chemical modification of the sugar part of methyl acarviosin: synthesis and inhibitory activities of nine analogues; Shibata Y et al.; Nine analogues of methyl acarviosin (1), the core structure of acarbose and its homologues, the 6-hydroxy-(2), 6-azido-(3), 6-amino- (4), 6-acetamido-(5), 6-methoxy-(6), 6-hydroxy-2-O-methyl-(8), and 6-hydroxy-3-O-methyl derivatives (9), including the 5-methoxycarbonyl analogue (7) and 3,6-anhydro derivative (10) of 2, were synthesized by chemical modification of the sugar part of 2 derived by condensation of methyl 3,4-anhydro-alpha-D-galactopyranoside (17) and 4,7:5,6-di-O-isopropylidenevalienamine (26) or by direct coupling between 26 and the 6-substituted methyl 3,4-anhydro-alpha-D-galactopyranoside derivatives . Compounds 2 and 8 show notable inhibitory activity against yeast alpha-D-glucosidase almost comparable to that of 1 . Introduction of a polar substituent at C-6 of 1 decreases the inhibitory activity . Interestingly, inversion of the conformation of the sugar part of 1 by introduction of the 3,6-anhydro bridge elicits almost no effect on the inhibitory activity.

Nucleic Acids Res, 1992 Apr 25, 20(8), 1903 - 8
Kinetic trapping of H-DNA by oligonucleotide binding; Belotserkovskii BP et al.; Homopurine-homopyrimidine mirror repeats are known to adopt the H form under acidic pH and/or negative supercoiling . In H-DNA, one half of the purine strand enters the triplex whereas the second half is unstructured and can form duplex with complementary oligonucleotide . However, because the same oligonucleotide can form triplex with the homopurine-homopyrimidine insert, one could expect that oligonucleotide would make H-DNA thermodynamically less favorable, as was claimed by Lyamichev et al . Nucl . Acids Res . 16, 2165-2178 (1988) . Now we show that complex between oligonucleotide and H-DNA, formed under conditions favorable for the H-form extrusion, is kinetically trapped in superhelical DNA and remains stable up much higher pH values than H-DNA alone . Experiments on chemical probing show that such complex exists for a plasmid with native superhelical density at pH7 . We have also used this approach to demonstrate a pH-dependent structural transition in yeast telomeric sequence, d(CACACCCA)16.

Nucleic Acids Res, 1992 Apr 25, 20(8), 1865 - 70
Efficient mischarging of a viral tRNA-like structure and aminoacylation of a minihelix containing a pseudoknot: histidinylation of turnip yellow mosaic virus RNA; Rudinger J et al.; Mischarging of the valine specific tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA has been tested in the presence of purified arginyl-, aspartyl-, histidinyl-, and phenylalanyl-tRNA synthetases from bakers' yeast . Important mischarging of a 264 nucleotide-long transcript was found with histidinyl-tRNA synthetase which can acylate this fragment up to a level of 25% with a loss of specificity (expressed as Vmax/KM ratios) of only 100 fold as compared to a yeast tRNA(His) transcript . Experiments on transcripts of various lengths indicate that the minimal valylatable fragment (n = 88) is the most efficient substrate for histidinyl-tRNA synthetase, with kinetic characteristics similar to those found for the control tRNA(His) transcript . Mutations in the anticodon or adjacent to the 3' CCA that severely affect the valylation capacity of the 264 nucleotide long TYMV fragment are without negative effect on its mischarging, and for some cases even improve its efficiency . A short fragment (n = 42) of the viral RNA containing the pseudoknot and corresponding to the amino acid accepting branch of the molecule is an efficient histidine acceptor.

J Biol Chem, 1992 Apr 25, 267(12), 8464 - 7
Mechanism of assembly of the RNA polymerase II preinitiation complex . Evidence for a functional interaction between the carboxyl-terminal domain of the largest subunit of RNA polymerase II and a high molecular mass form of the TATA factor; Conaway RC et al.; Genetic evidence argues that the highly conserved carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions directly in the regulation of transcription of many eukaryotic genes . The observation that partial deletion of the CTD of yeast RNA polymerase II reduces the ability of the enzyme to respond to signals from a variety of upstream activating sequences led to the proposal that the CTD plays a role in the dialogue between regulatory factors that bind upstream activating sequences and the "general" or "basal" transcription factors associated with RNA polymerase II at the promoter (Scafe, C., Chao, D., Lopes, J., Hirsch, J . P., Henry, S., and Young, R . A . (1990) Nature 347, 491-494) . Biochemical evidence for an interaction of the CTD with specific components of the basal transcription apparatus, however, has been lacking . To identify target(s) for CTD action, we probed steps in assembly of the RNA polymerase II preinitiation complex with monoclonal antibodies specific for the CTD . Our findings reveal a novel interaction of the CTD with a high molecular mass form of the TATA factor . This interaction occurs during binding of RNA polymerase II to its promoter and requires the action of additional basal transcription factors; it is not observed when the single-subunit yeast transcription factor IID serves as the TATA factor.

J Biol Chem, 1992 Apr 25, 267(12), 8286 - 92
Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor . A DNA-binding domain and a subunit interaction domain are specified by two separate segments; Maity SN et al.; CCAAT-binding factors A (CBF-A) and B (CBF-B) are two subunits of the heteromeric CCAAT-binding factor . Portions of CBF-A and CBF-B have a high degree of amino acid sequence identity to segments of the HAP3 and HAP2 subunits of a yeast multimeric transcription factor . We show here that the subunits of CBF interact with each other in the absence of DNA binding . This interaction was revealed by cross-linking and coimmunoprecipitation studies . Both the DNA binding and subunit interaction functions of CBF-B have been examined by mutational analysis . A segment of 83 amino acids from residues 252 to 334, which corresponds to the evolutionarily conserved portion of CBF-B, is necessary and sufficient for CBF-A-dependent DNA binding . Carboxyl-terminal deletions of this segment (or mutations in arginine residues in this carboxyl-terminal part) abolish DNA binding, but do not alter subunit interactions between CBF-A and CBF-B . Mutations in hydrophobic amino acids within the amino-terminal part of the evolutionarily conserved sequence at positions 252-334 result in loss of both DNA binding and subunit interaction activities . Our results indicate that the evolutionarily conserved segment of CBF-B contains both DNA-binding and subunit interaction domains and that the integrity of both domains is essential for DNA binding.

J Biol Chem, 1992 Apr 25, 267(12), 8270 - 4
Sequence requirements for precursor cleavage within the constitutive secretory pathway; Watanabe T et al.; We have recently demonstrated that the Arg-X-Lys/Arg-Arg sequence is a signal for precursor cleavage catalyzed by furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, within the constitutive secretory pathway . In this study, we further examined sequence requirements for the constitutive precursor cleavage by expression of various prorenin mutants with amino acid substitutions around the native Lys-Arg cleavage site in Chinese hamster ovary cells . The results delineate the following sequence rules that govern the constitutive precursor cleavage . (a) A basic residue (Lys or Arg) at the 4th (position -4) or 6th (position -6) residue upstream of the cleavage site besides basic residues at positions -1 and -2 is necessary . (b) At position -2, a Lys residue is more preferable than Arg . (c) At position -4, an Arg residue is more preferable than Lys . (d) At position 1, a hydrophobic aliphatic amino acid is not suitable.

J Biol Chem, 1992 Apr 25, 267(12), 7979 - 82
Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases; Mu D et al.; The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S . M., Mu, D., Wemmer, D., Smith, A . J., Kaur, S., Maltby, D., Burlingame, A . L., and Klinman, J . P . (1990) Science 248, 981-987) . The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P . G., Evers, M., Waterham, H . R., Kuipers, J., Arnberg, A . C., and Geert, A . B . (1989) Biochim . Biophys . Acta 1008, 157-167) . In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H . polymorpha . Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with {14C}phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography . Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor . By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein . In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.

J Biol Chem, 1992 Apr 25, 267(12), 8679 - 84
Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptide from mammalian brain; Bureau MH et al.; A polypeptide of M(r) 36,000 (36 kDa) was isolated from detergent-solubilized membrane fractions of mammalian brain on a benzodiazepine affinity column utilized for the purification of the gamma-aminobutyric acid/benzodiazepine receptor protein, followed by preparative gel electrophoresis . Partial protein sequence for two fragments of the 36-kDa polypeptide allowed the isolation of cDNA clones from a rat hippocampal library . An open reading frame coding a sequence of 295 amino acid residues containing the two probe peptide sequences with minor differences, and a putative N-terminal signal peptide of 25 residues was found . Hydropathy index revealed no regions of alpha-helix suitable for membrane spanning, but several areas of alternating hydrophilic and hydrophobic residues consistent with beta-strands . The sequence of this brain protein was 24% identical to that of a yeast mitochondrial protein, the voltage-dependent anion channel (VDAC), and over 70% identical with the VDAC from human B lymphocytes . The gamma-aminobutyric acid type A (GABAA) receptor/36-kDa preparation purified on benzodiazepine affinity column has channel-forming activity in lipid bilayer membranes that is virtually identical to VDAC isolated from mitochondria of various sources, indicating that the 36-kDa protein is a new member of the VDAC family of proteins . An antiserum raised against the purified 36-kDa polypeptide was able to precipitate {3H}muscimol binding activity, indicating a tight association with the GABAA receptor protein in vitro and copurification on the benzodiazepine affinity column due to this association . Further studies are needed to determine whether such an association occurs in vivo.

J Biol Chem, 1992 Apr 15, 267(11), 7904 - 10
Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase; Isaya G et al.; We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus . We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins . Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity . MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition . To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP . The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate . Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein . When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta . Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.

Eur J Biochem, 1992 Apr 15, 205(2), 537 - 43
M-phase-specific histone H1 kinase in fish oocytes . Purification, components and biochemical properties; Yamashita M et al.; We demonstrate, for the first time in fish, that a Ca(2+)-independent and cyclic-nucleotide-independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them . The kinase, M-phase-specific histone H1 kinase (M-H1K), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPKKAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serine/threonine-specific protein kinase encoded by the fission yeast cdc2+ gene (cdc 2 kinase) . The M-H1K and maturation-promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M-H1K and MPF . The final preparation was purified 5000-fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10,000-fold with a recovery of 7% when SP peptide was used . The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33, 34, 46 and 48 kDa . Anti-PSTAIR antibody recognizing cdc2 kinase cross-reacted with the 33-kDa and 34-kDa proteins, while the 46-kDa and 48-kDa bands cross-reacted with monoclonal antibodies raised against cyclin B . The 33-kDa protein was also recognized by an antibody against a goldfish cdk2 (Eg1) kinase, a cdc2-related kinase which has the PSTAIR sequence and binds to p13suc1 but does not form a complex with cyclin B . M-H1K activity corresponded well to the 34-kDa, 46-kDa and 48-kDa proteins but not to the 33-kDa protein . These results strongly suggest that M-H1K consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M-H1K, although it is found in the highly purified M-H1K . The purified M-H1K utilized Mg2+, Mn2+, ATP and GTP, and had a wide pH optimum ranging over 8.0-10.5 . The kinase was thermolabile and sensitive to freezing/thawing.

FEBS Lett, 1992 Apr 13, 301(1), 23 - 8
Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme; el Dirani-Diab R et al.; The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions . A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized . The enzyme was affinity labeled using a 5' modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor . We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form . Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT forms . The effect of primer tRNA(Lys) on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA(Lys), like RT p66/p66, when KCl was present in the incubation mixture.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1457 - 62
DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts; Mahbubani HM et al.; Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control . We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system . Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin . 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp) . Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule . Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.

Biochemistry, 1992 Apr 7, 31(13), 3472 - 7
Photoinduced electron transfer between cytochrome c peroxidase and horse cytochrome c labeled at specific lysines with (dicarboxybipyridine)(bisbipyridine)ruthenium(II)
Hahm S, Durham B, Millett F.
The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) {Ru(II)} were studied by flash photolysis . All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7) . Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI . This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI . The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively . The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives . These results suggest that there are two binding orientations for cytochrome c on CMPI . The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer . Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase . A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength . When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed . The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.

Cell, 1992 Apr 3, 69(1), 173 - 84
chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis; Cooley L et al.; The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis . chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport . We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin . The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly . In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm . We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells.

Int J Immunopharmacol, 1992 Apr, 14(3), 391 - 7
Pre-clinical toxicity of IL-4: a model for studying protein therapeutics; Dean JH et al.; The purpose of this presentation was to review issues and findings in the pre-clinical development and evaluation of recombinant human protein therapeutics . Since human cytokines and lymphokines are endogenous proteins, their pre-clinical development and evaluation would seem straightforward and their toxicities minimal . Unfortunately, the pre-clinical development of this class of agents has been problematic and confounding . Some of the clinical toxicities and pharmacodynamics have been predicted by the pre-clinical evaluation and others have not . Some molecules are species specific which limits species selection for pre-clinical evaluation . Other confounding issues include: route of exposure, synergy of toxicity with other lymphokines, length of study design, immunogenicity, predictiveness of pre-clinical evaluation and iatrogenic toxicities . An approach used by SWPRD in the evaluation of this class of molecules was discussed . Insight gained during the pre-clinical and clinical development of these molecules should simplify the further development of protein therapeutics that follow . Specific studies with recombinant human interleukin-4 (rhuIL-4) were reviewed in detail as part of a pre-clinical safety evaluation . Native IL-4 has properties that exemplify many of the immune recognition-induced lymphokines and is produced principally by activated T-lymphocytes CD4+ . It is a co-factor in B-cell proliferation and enhances ex vivo B-cell expansion and is believed to be a candidate for the treatment of refractory cancer based on this immune enhancement ability . rhuIL-4 is a 15,400 molecular weight cytokine produced in a yeast expression system.(ABSTRACT TRUNCATED AT 250 WORDS)

Inflammation, 1992 Apr, 16(2), 83 - 91
Phagocytosis by lipopolysaccharide-primed human neutrophils is associated with increased extracellular release of reactive oxygen metabolites; Follin P et al.; The effect of priming human neutrophils with lipopolysaccharide was investigated regarding the respiratory burst activity generated during phagocytosis of IgG- or C3b-opsonized yeast particles . LPS pretreatment significantly enhanced the respiratory burst activity, measured as luminol-amplified chemiluminescence, of both types of opsonized particles . In control cells most of the activity was produced intracellularly, probably in the phagosomes . In the primed cells, however, extracellular release of reactive oxygen metabolites was significantly increased during Fc- and CR3-mediated phagocytosis (P less than 0.01 and P less than 0.002, respectively) . The release was most pronounced when using C3b-opsonized particles . Potent oxygen metabolites acting together with lysosomal enzymes are of importance in inflammatory-induced tissue damage . An increased extracellular release of reactive oxygen species by phagocytizing primed neutrophils can therefore lead to greater damage to the surrounding tissues.

Comput Appl Biosci, 1992 Apr, 8(2), 177 - 84
DNA Translator and Aligner: HyperCard utilities to aid phylogenetic analysis of molecules; Eernisse DJ; DNA Translator and Aligner are molecular phylogenetics HyperCard stacks for Macintosh computers . They manipulate sequence data to provide graphical gene mapping, conversions, translations and manual multiple-sequence alignment editing . DNA Translator is able to convert documented GenBank or EMBL documented sequences into linearized, rescalable gene maps whose gene sequences are extractable by clicking on the corresponding map button or by selection from a scrolling list . Provided gene maps, complete with extractable sequences, consist of nine metazoan, one yeast, and one ciliate mitochondrial DNAs and three green plant chloroplast DNAs . Single or multiple sequences can be manipulated to aid in phylogenetic analysis . Sequences can be translated between nucleic acids and proteins in either direction with flexible support of alternate genetic codes and ambiguous nucleotide symbols . Multiple aligned sequence output from diverse sources can be converted to Nexus, Hennig86 or PHYLIP format for subsequent phylogenetic analysis . Input or output alignments can be examined with Aligner, a convenient accessory stack included in the DNA Translator package . Aligner is an editor for the manual alignment of up to 100 sequences that toggles between display of matched characters and normal unmatched sequences . DNA Translator also generates graphic displays of amino acid coding and codon usage frequency relative to all other, or only synonymous, codons for approximately 70 select organism-organelle combinations . Codon usage data is compatible with spreadsheet or UWGCG formats for incorporation of additional molecules of interest . The complete package is available via anonymous ftp and is free for non-commercial uses.

J Interferon Res, 1992 Apr, 12(2), 119 - 29
Molecular cloning of porcine Mx cDNAs: new members of a family of interferon-inducible proteins with homology to GTP-binding proteins; Muller M et al.; Porcine cells treated with interferon (IFN) or double-stranded RNA synthesize two proteins that exhibit high homology of the amino acid sequence to mouse Mx1 protein involved in selective resistance to influenza virus . A full-length cDNA clone (poMx1) encoding the porcine Mx1 protein was isolated and sequenced . It contained an open reading frame of 663 amino acids . The predicted molecular weight of 75.6 kD is in good agreement with the apparent molecular mass of the two immunoprecipitable proteins of 76 kD and 73 kD determined by SDS polyacrylamide gel electrophoresis . A second cDNA (poMx2) was characterized which was incomplete in the 5' region . A comparison of all known Mx proteins revealed an average homology of 67.5% . The porcine Mx1 polypeptide is most closely related to human MxA (p78), murine Mx2, rat Mx2, and rat Mx3 proteins . The amino-terminal halves of all Mx proteins are highly conserved and possess three consensus elements in proper spacing, characteristic of GTP-binding domains . The Mx family shows in their amino termini striking homology to previously characterized Mx-related proteins playing roles in the intracellular vectorial transport of proteins--the products of the yeast Vps1 locus and the dynamins.

Carcinogenesis, 1992 Apr, 13(4), 609 - 15
Reversion of the hprt mutant clone SP5 by intrachromosomal recombination; Zhang LH et al.; The spontaneous hprt mutant clone SP5, derived from V79 Chinese hamster cells, was shown to exhibit a duplication of approximately 2 kb, including exon 2 and its flanking intron sequences, inserted into the intron 1 sequence of the hprt gene . The most striking feature of SP5 is that this clone is quite unstable, demonstrating an extremely high spontaneous reversion frequency . Molecular analysis of 25 independent revertant clones of SP5 indicated that they arose after precise deletion of the duplicated fragment in the hprt gene . Reversion of SP5 could be induced by agents which damage DNA by different mechanisms, but there was no correlation with induction of the forward mutations . Based on these results, we suggest that intrachromosomal recombination must be responsible for the spontaneous reversion of SP5 . Genetic recombination in somatic cells has been suggested to be involved in the multistep process of carcinogenesis . Since the ability to induce intrachromosomal recombination in yeast has been shown to be highly correlated with non-mutagenic as well as mutagenic carcinogens, it is of great interest to investigate similar systems in mammalian cells . The SP5 cell line may be unique for such a purpose, since this mutant clone contains an endogenic marker for studying the process of intrachromosomal recombination.

Eur J Biochem, 1992 Apr 1, 205(1), 211 - 6
Molecular characterization of a Leishmania donovani infantum antigen identified as histone H2A; Soto M et al.; A Leishmania donovani infantum promastigote cDNA expression library was screened with a serum obtained from a dog naturally infected with this parasite . One of the positive clones obtained revealed nucleotide sequence similarities with the histone H2A genes from various organisms . Northern blot analyses and sequence data of three independently isolated cDNA clones indicated that the Leishmania H2A mRNAs are polyadenylated, as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast . The analysis of the genomic distribution of the DNA coding for histone H2A suggested that, in L . d . infantum, there are at least four genes coding for the H2A protein . It is likely that there is a simultaneous expression of at least two of the H2A genes since differences in nucleotide sequence between two of the sequenced cDNAs were observed . Affinity-purified antibodies against the beta-galactosidase-fused H2A protein recognize specifically a Leishmania protein band with a molecular mass of 14 kDa.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1992 Apr, 14(2), 155 - 8
{An approach to the preparation of digestive enzyme-drug for oral administration: enteric-soluble disaccharidase microcapsules}; Wang X; For the purpose of replacement therapy in cases of disaccharide intolerances, yeast disaccharidases were prepared as enteric-soluble microcapsules to prevent their inactivation by gastric juice and alimentary proteases . The enteric-soluble microcapsules have a mean diameter of 0.544 +/- 0.058 mm . Following encapsulation, activity recoveries for invertase and lactase were 84.4 +/- 10.6% and 70.1 +/- 5.5%, respectively . Over 55% of the remaining microcapsulated disaccharidase activity was retained in artificial gastric juice during 2 h of coincubation . Over 90% of the enzyme activity was released from the microcapsules within 1 h in artificial intestinal juice.

Curr Opin Cell Biol, 1992 Apr, 4(2), 144 - 8
Cell proliferation and control; Pines J; In the past year much of the focus in cell-cycle research has turned from the regulation of mitosis to the control of the initiation of DNA replication . Novel findings include the discovery of vertebrate G1 cyclins, an additional cdc2-related kinase potentially involved in G1 phase, and a positive-feedback loop regulating the start of the cell cycle in yeast.

Lab Invest, 1992 Apr, 66(4), 498 - 503
Monocyte chemoattractant protein 1 in a rat model of pulmonary granulomatosis; Jones ML et al.; Monocyte chemoattractant protein 1 (MCP1), also known as monocyte chemotactic and activating factor, possesses potent chemotactic activity for monocytes and can augment monocyte tumoristatic activity against some tumor cell lines . While these activities suggest a role in inflammatory and immunologic processes, the biologic role of MCP1 has not been studied in vivo . Glucan-induced pulmonary granulomatosis in the rat is an ideal model in which to study the role of MCP1 because the granulomas are monocyte/macrophage rich . Intravenous infusion of particulate yeast cell wall glucan resulted in the synchronous development of angiocentric pulmonary granulomas . Early lesions (6 hours) were characterized by intravascular glucan aggregates surrounded by neutrophils and foci of alveolar hemorrhage while later appearing granulomatous lesions (48 to 96 hours) were dominated by monocytes and macrophages . Granuloma formation was paralleled by a peripheral blood monocytosis . Analysis of bronchoalveolar lavage (BAL) fluid revealed an early, transient rise in tumor necrosis factor activity followed by a marked rise in monocyte-specific chemotactic activity . The rise in BAL fluid monocyte chemotactic activity, which coincided with the development of the monocyte/macrophage-rich granulomas, was preceded by a marked increase in whole lung MCP1 mRNA expression . BAL fluid monocyte chemotactic activity could be nearly completely neutralized with antibody directed against rat MCP1 . These studies demonstrate that MCP1 mRNA expression is upregulated in glucan-induced pulmonary granulomatosis and that MCP1 is present in BAL fluid . Intrapulmonary granulomatosis may be important in the pathogenesis of granuloma formation.

J Clin Invest, 1992 Apr, 89(4), 1172 - 7
Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core . Implications for paroxysmal nocturnal hemoglobinuria; Thomas LJ et al.; A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized . The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level . We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level . The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway . Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants . These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences . Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Mol Biol Cell, 1992 Apr, 3(4), 403 - 14
SpCoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury; Smith LC et al.; SpCoel1 is a single copy gene that is specifically expressed in most of the coelomocytes of the adult purple sea urchin, Strongylocentrotus purpuratus . The 4-kb transcript from this gene has a relatively short (426 nucleotide) open reading frame (ORF) with long 3' and 5' untranslated regions . The ORF encodes a protein that has strong amino acid sequence similarity to profilins from yeast to mammals . Transcript titrations of SpCoel1 show significant increases per coelomocyte in animals that have been physiologically challenged . Increases in transcript levels are of similar magnitudes between animals receiving different treatments, such as injuries from needle punctures or from injections of foreign cells . The evidence presented here implies a molecular mechanism by which this lower deuterostome defense system responds to external insult, viz that an external "injury signal" activates a signal transduction system, which in turn mediates the alterations in cytoskeletal state that are required for coelomocyte activation.

Mycopathologia, 1992 Apr, 118(1), 29 - 36
Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi; el-Sayed GN et al.; An apiZYM system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae . Similar enzyme profiles were obtained for two of the N . rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates . Centroid-clustering analysis revealed three distinct enzyme profiles.

Biol Trace Elem Res, 1992 Apr-Jun, 33, 197 - 204
Selenium content and distribution in rat tissues irradiated with gamma rays; Djujic IS et al.; The effects of supplementation with selenous yeast and ionizing radiation on selenium (Se) content and distribution were evaluated in rat tissues (liver, kidney, spleen, heart, muscle, blood, front brain, hind brain, hypothalamus, pituitary, adrenal glands, testes, and hair) . This study had 16 Se-supplemented (0.5 micrograms Se/d) and 16 placebo adult male Wistar rats . One half of the animals (eight Se-supplemented and eight placebos) were irradiated with a single dose of 4.2 Gy from a Co-60 source and sacrificed 7 d after irradiation along with nonirradiated animals and analyzed for Se content determination . The data obtained showed that selenous yeast supplementation increased Se levels in rat tissues (highest increases in hypothalamus, 161%; hind brain, 126%; spleen, 110%; and adrenal gland, 105%) . Ionizing radiation induced significant changes in Se content and distribution (decrease in liver, blood, hair, femoral muscle, spleen, and hypothalamus; increase in kidney, testes, adrenal glands, and brain of placebo group) . Supplementation with selenous yeast reduces changes in Se content and distribution after irradiation . It seems that the animal tissue susceptibility to oxidative damage may be correlated to their ability to retain Se in tissues.

Appl Microbiol Biotechnol, 1992 Apr, 37(1), 109 - 13
Comparison of the catalytic and inhibitory properties of Pachysolen tannophilus xylose reductase to rat lens aldose reductase; Davis RA et al.; The catalytic and inhibitory profiles of xylose reductase isolated from the yeast Pachysolen tannophilus (PTXR) are compared to those of aldose reductase (AR) obtained from rat lens . While both PTXR and rat lens AR are NADPH-specific enzymes and have an affinity for a variety of substrates such as D-xylose, D,L-glyceraldehyde, and 4-nitrobenzaldehyde, the enzymes differ in their substrate affinity profiles . Also, PTXR is not inhibited by standard inhibitors of AR thus supporting a hypothesis that this enzyme may not possess the inhibitor binding site found in rat lens AR.

Hum Genet, 1992 Apr, 89(1), 33 - 6
Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis; Boretto J et al.; Two yeast artificial chromosomes of the HLA class I region were subcloned . Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series . Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis . Only one marker presents an unequivocal association with the haemochromatosis gene and is 100 kb centromeric to HLA-A . This association however is not as strong as with HLA-A3 . The results suggest two possible locations for the haemochromatosis gene: less than 100 kb centromeric to the HLA-A locus, or on the telomeric side.

Glycoconj J, 1992 Apr, 9(2), 75 - 81
O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii; Lopes-Alves LM et al.; beta-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases of Sporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine . Man-(alpha 1-2)Man-ol, Rha(alpha 1-3)Man(alpha 1-2)Man-ol, Rha(alpha 1-4)GlcA(alpha 1-2)Man(alpha 1-2)Man-ol, and Rha(alpha 1-4){Rha(alpha 1-2)} GlcA(alpha 1-2)Man(alpha 1-2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.

Brain Pathol, 1992 Apr, 2(2), 121 - 32
Defects of mitochondrial DNA; Zeviani M et al.; In the past few years several syndromes have been associated with lesions of the human mitochondrial DNA . MtDNA is a small, circular extra-nuclear chromosome encoding essential components of the respiratory chain . MtDNA-related syndromes can be divided into two groups: mitochondrial encephalomyopathies, characterized by the presence of ragged-red fibres (RRF) as the morphological hallmark, or "pure" encephalopathies with no gross morphological abnormalities in muscle . The first group includes myoclonic epilepsy with ragged-red fibres (MERRF), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO) and a new entity, maternally inherited myopathy and cardiomyopathy . The second group includes Leber's Hereditary Optic Neuroretinopathy (LHON) and the newly described ataxia-retinitis pigmentosa-dementia complex . Three kinds of molecular lesions have been identified: point mutations of protein encoding mtDNA-genes (similar to yeast mit- mutations); point mutations of mtDNA-tRNA genes (similar to yeast syn- mutations); and large-scale rearrangements of mtDNA (similar to yeast rho- mutations) . In general, "mit-" mutations are responsible for non-RRF encephalopathies, while "syn-" and "rho-" mutations are associated with mitochondrial encephalomyopathies with RRF . Furthermore, point mutations (mit- and syn-) are usually maternally- inherited, while large-scale mtDNA rearrangements are either sporadic or inherited as mendelian traits . In most cases, the molecular detection of the known defects of mtDNA can be carried out by non-invasive techniques, thus making it an easy and relatively inexpensive procedure in the differential diagnosis of the mitochondrial disorders, a rapidly expanding area of clinical neurology.

Hum Mol Genet, 1992 Apr, 1(1), 3 - 6
Beginning or end? Telomere structure, genetics and biology; Kipling D et al.; The word telomere derives from the Greek word telos meaning 'end', roughly translating as 'the thing at the end' when the end is that of a chromosome . Telomeres belie their apparent simplicity of structure by being involved in a wide range of diverse biological phenomena . Much of our understanding of telomere behaviour comes from studies in lower eukaryotes such as ciliates and yeast, the subject of many recent reviews . Here we concentrate on the mammalian telomere, recent progress in its study, and how recent evidence for an involvement of telomeres in the regulation of gene expression and DNA replication in yeast points to new aspects of mammalian telomere function yet to be explored.

Nature, 1992 Mar 26, 356(6367), 353 - 5
The wee1 protein kinase is required for radiation-induced mitotic delay; Rowley R et al.; Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions . In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage . Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis . Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements . We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway . Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1.

Eur J Obstet Gynecol Reprod Biol, 1992 Mar 23, 44(1), 77 - 80
Vulvovaginal candidiasis refractory to treatment with fluconazole; Arilla MC et al.; We present the case of an infertile patient, whose first attempt at IVF had to be postponed for 18 months due to a vulvovaginal yeast infection refractory to treatment . The main causative organism was a Candida glabrata strain resistant to all the imidazolic agents tested . The organism and the host's humoral status were studied in depth, looking for possible causes of the refractoriness to treatment.

J Mol Biol, 1992 Mar 20, 224(2), 343 - 58
Localization and DNA sequence of a replication origin in the rhodopsin gene locus of Chinese hamster cells; Gale JM et al.; A chromosomal origin of DNA replication has been localized within the single-copy rhodopsin gene locus in Chinese hamster (line CHO) cells using two methods . In the first method, single-copy segments were identified at 3 to 15 kb intervals within approximately 75 kb (kb = 10(3) bases) of cloned genomic DNA containing the early-replicating rhodopsin gene near its middle . The cloned single-copy segments were then used as hybridization probes to quantify the replication of their corresponding genomic segments as synchronized cells progressed into S phase . In the second method, genomic DNA synthesized in vivo or in permeabilized early S phase cells was hybridized with slot-blots of the cloned single-copy DNA segments to identify the earliest replicating part of the 75 kb mapped region . The first method indicates that the earliest replicating DNA is located within a 10 kb region beginning 4 kb upstream from and extending 1 kb beyond the rhodopsin gene . The second method confirms the location in the vicinity of the rhodopsin gene and indicates that the earliest replicating region is located within or very near the 4.5 kb rhodopsin gene itself . An extended region of 12 kb that encompasses the entire early-replicating region has been sequenced for analysis and comparison with currently characterized origin regions associated with the CHO dihydrofolate reductase (dhfr) and human c-myc genes . There are several sequence similarities between the dhfr rhodopsin origin regions, including common transcription promoter consensus sequences, rodent Alu repeats with their 3'-A+T rich flanking sequences, A+T-rich yeast ARS and Drosophila SAR consensus sequences, and simple (GA)n repeats, but there are no extended regions of direct similarity . The rhodopsin gene locus is the second sequenced CHO origin region.

Biochem Pharmacol, 1992 Mar 17, 43(6), 1345 - 51
Mechanisms of inactivation of Schistosoma mansoni and mammalian glutathione S-transferase activity by the antischistosomal drug oltipraz; Nare B et al.; Glutathione S-transferase (GST) purified from Schistosoma mansoni or human placenta was inhibited by the antischistosomal drug oltipraz (OPZ) in a time- and concentration-dependent manner . Inhibition of placenta GST was complete at a low concentration of drug, whereas that of parasite GST was incomplete and relatively high amounts of OPZ were needed to reach 50% inhibition . Complete reactivation of GST from placenta was achieved with dithiothreitol (DTT) and other sulfhydryl-containing compounds, while the inactivation of parasite GST was irreversible . The oxy-derivative of OPZ (RP 36,642), in which the thione sulfur is replaced with oxygen, did not inhibit GST activity . There were no differences between OPZ and RP 36,642 in their patterns of binding to the hydrophobic non-substrate site of GST . GST from the placenta incorporated much higher levels of {14C}N-ethylmaleimide compared to schistosome GST . The incorporation of {14C}N-ethylmaleimide by GST was inhibited by OPZ but not by RP 36,642 . Yeast and S . mansoni hexokinases were similarly inhibited by OPZ but not by RP 36,642 . Both hexokinase preparations recovered their activity following incubation with DTT . These data suggest that the inactivation of these enzymes by OPZ is a result of its interaction with their SH groups . Thus, the antischistosomal activity of OPZ may be accounted for by its interaction with the SH groups of macromolecules in general.

Carbohydr Res, 1992 Mar 16, 226(1), 79 - 89
Synthesis and enzyme-inhibitory activity of methyl acarviosin analogues having the alpha-manno configuration; Ogawa S et al.; Two methyl acarviosin analogues 3a and 4a, having the alpha-manno configuration, and their dihydro derivatives 6a and 7a were synthesised by coupling the protected pseudo-sugar epoxides with methyl 4-amino-4-deoxy- and -4,6-dideoxy-alpha-D-mannopyranoside . Similarly, two analogous compounds 5a and 8a composed of the 1,6-anhydro-beta-D-mannopyranose residues were prepared . Compound 7a showed mild inhibitory activity against Jack bean alpha-D-mannosidase, and 3a was a moderate inhibitor of both alpha-D-mannosidase and yeast alpha-D-glucosidase.

J Immunol, 1992 Mar 15, 148(6), 1804 - 11
Regulation of synthesis of p34cdc2 and its homologues and their relationship to p110Rb phosphorylation during cell cycle progression of normal human T cells; Lucas JJ et al.; In yeast, the protein kinase p34cdc2 plays a role in regulating both the G2 to M and G1 to S phase transitions . The discovery of multiple homologues of the protein in cells of higher eukaryotic organisms suggests that different cell cycle regulatory events may be performed by different kinases in such cells . Here, the synthesis and metabolism of the human forms of these proteins are described in a normal human cell type, peripheral blood T lymphocytes that have been stimulated to enter the cell cycle in vitro . Using a carboxyl-terminus antiserum specific for true p34cdc2, the protein could first be found in T cells at about 24 to 30 h after stimulation, just before the initiation of DNA synthesis . Three forms of the enzyme could be resolved by denaturing gel electrophoresis: an unphosphorylated form with an apparent molecular mass of 34,500 daltons and two phosphorylated derivatives . In cells synchronized at G2/M phase with nocodazole, p34 was almost entirely in the unphosphorylated form whereas the phosphorylated derivatives were more predominant in cultures arrested at the G1/S border with aphidicolin . The relationship of p34 synthesis to the phosphorylation of p110Rb, an event known to be associated with passage through late G1 and/or the G1/S phase transition, was also investigated . It was noted that p110Rb phosphorylation began before p34 synthesis first became detectable . Furthermore, it appeared that the two events could be largely uncoupled by treating cells with deferoxamine (10 microM), an iron chelating agent that arrests T cells at a point in late G1 phase but substantially before the G1 to S phase transition . Under these conditions, p110Rb phosphorylation was almost completely accomplished in the absence of significant p34 synthesis, a finding that suggests that most or all of p110 phosphorylation is performed by kinases other than p34 . Because of this observation, extracts were next examined for p34-like molecules using an antibody against the so-called PSTAIRE domain found in all cdc2 homologues identified to date . A species of protein with a mobility slightly less than true p34 was found, even in resting T cells . Upon stimulation, this protein increased slightly in amount, and a second protein with a mobility greater than p34, a putative p33cdk2, was seen . Not only was the appearance of these proteins not inhibited by deferoxamine but they accumulated in cultures treated with the drug, suggesting that p33, and not p34, may be the G1 phase kinase for p110Rb.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene, 1992 Mar 15, 112(2), 147 - 55
Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase; Kuijper JL et al.; This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system . The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA . This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA . The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA . The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system . The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers . The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners . This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization.

J Biol Chem, 1992 Mar 15, 267(8), 5676 - 9
Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis; Imamura T et al.; We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1) . The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1 . In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells . Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus . Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants . These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro.

Science, 1992 Mar 13, 255(5050), 1404 - 8
Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease; Baldi MI et al.; Introns have generally been assumed to be passive in the transfer RNA splicing reaction . Experiments have now been done showing that the endonuclease is able to cut a precursor provided that a base in the single-stranded loop of the intron can pair with the base of the 5' exon situated at the position that immediately follows the anticodon stem (position 33 in the yeast tRNA isoacceptor pre-tRNA(Leu)3, position 32 in yeast pre-tRNA(Phe)) . The elucidation of the role of the intron reveals that in addition to the conserved bases, there are positions in the mature domain which, although not necessarily occupied by the same base in all pre-tRNA's, nevertheless have a fundamental role in the splicing reaction . These positions are termed cardinal positions.

Nature, 1992 Mar 12, 356(6365), 159 - 61
Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits; Federman AD et al.; Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits . In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively . By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways . Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma . This effect of beta gamma requires costimulation by alpha s-GTP . This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active . We now confirm this prediction . Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis . Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation . These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.

J Virol, 1992 Mar, 66(3), 1389 - 93
LRV1 viral particles in Leishmania guyanensis contain double-stranded or single-stranded RNA; Weeks R et al.; The 32-nm-diameter spherical viral particles found in the cytoplasm of Leishmania guyanensis CUMC1-1A sediment at 130S and have a buoyant density of approximately 1.4 g/ml in cesium chloride gradients . These particles contain a 5.3-kb double-stranded RNA, while single-stranded RNA that corresponds to the viral positive strand is associated with less-dense particles . These results suggest a conservative and sequential mode of LRV1 viral RNA replication that is exemplified by the ScV L-A virus of yeast.

Nucleic Acids Res, 1992 Mar 11, 20(5), 1087 - 91
Evolutionary variation of the CCAAT-binding transcription factor NF-Y; Li XY et al.; NF-Y is a CCAAT-specific transcription factor thought to be involved in the regulation of a variety of eukaryotic genes . It shows a striking sequence similarity with the yeast factor HAP2/3 . In an attempt to trace back its evolutionary history, we succeeded in isolating NF-Y cDNA clones from a plant and from several species of vertebrates . The patterns of sequence conservation delineate potential functional domains: A central, highly conserved, domain likely responsible for DNA-binding and subunit interaction; more evolutionarily flexible flanking regions, in which variability is clustered, individualizing conserved glutamine or acidic amino-acids putatively involved in protein-protein contacts.

Biochim Biophys Acta, 1992 Mar 5, 1116(1), 67 - 71
Formation of beta-galactosyl compounds of pyridoxine in growing culture of Sporobolomyces singularis; Suzuki Y et al.; Several pyridoxine compounds were found to be formed in a high yield in a growing culture of Sporobolomyces singularis containing lactose and pyridoxine . Three compounds, I, II and III, were isolated from cultured broth by Dowex 50W-X8 column chromatography, paper chromatography, lyophilization, and then obtained as needle crystals (m.p . (decomp.): I, 204-206 degrees C; II, 192-194 degrees C; III, 222-223 degrees C . {alpha}D16: I, -27.7 degrees (c = 3.82, H2O); II, -1.6 degrees (c = 2.52, H2O); III, +8.3 degrees (c = 3.98, H2O)) . Compounds I, II and III were identified as 5'-O-(beta-D-galactopyranosyl)-pyridoxine, 4'-O-(beta-D-galactopyranosyl)-pyridoxine, and 4'-O-(beta-D-galactopyranosyl-(1----4)-beta-D-galactopyranosyl)-pyrid oxi ne, respectively, on the basis of the various experimental results, viz., ultraviolet, infra-red, 1H-NMR, and 13C-NMR spectra, products by hydrolysis with acid and with alpha- and beta-galactosidases, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid . Also, the yeast produced a remarkable amount of beta-glucosyl compounds of pyridoxine in cultured broth, when grown on cellobiose and pyridoxine.

Nature, 1992 Mar 5, 356(6364), 80 - 3
Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation; Joshi HC et al.; gamma-Tubulin is a newly identified member of the tubulin family whose sequence is highly conserved from yeast to man . This minor microtubule protein is localized to the microtubule organizing centres and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans . Here we investigate the in vivo function of gamma-tubulin in mammalian cells using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of gamma-tubulin . After microinjection into cultured mammalian cells, immunofluorescence localization revealed that this antibody binds to native centrosomes at all phases of the cell cycle . In the presence of the gamma-tubulin antibody, microtubules fail to regrow into cytoplasmic arrays after depolymerization induced by nocodazole or cold . Furthermore, cells injected immediately before or during mitosis fail to assemble a functional spindle . Thus in vivo gamma-tubulin is required for microtubule nucleation throughout the mammalian cell cycle.

Eur J Epidemiol, 1992 Mar, 8(2), 305 - 8
Hansenula anomala fungemia in an infant with gastric and cardiac complications with a review of the literature; Sekhon AS et al.; A 6-month-old female infant, with a birth weight of 2.74 kilograms, was born with multiple congenital abnormalities, including gastric and gastrointestinal defects . She was admitted to the hospital with hematemesis . The patient could not be fed orally, and parenteral nutrition was initiated through a central venous catheter . Following pyloroplasty, she developed superior vena cava syndrome, renal disfunction and episodes of sepsis . Stool and respiratory specimens were negative for fungi, but four blood cultures yielded Hansenula anomala var . anomala . Cultures for fungi from intravenous catheter tips were negative . The baby was treated with amphotericin B (am B) and 5-fluorocytosine (5-FC), (amB; 0.1 mg/kg body weight and 5-FC, 100 mg, q.i.d.) . The minimal inhibitory concentrations of am B, 5-FC, am B + 5-FC (1:1, w:w) and fluconazole to H . anomala were 1.56, less than 0.195, 1.56, and 1.56 micrograms, respectively . Following antifungal therapy and removal of the catheter, the patient tolerated oral feeding and, at the time of discharge, her weight had increased to 4.91 kg . This report records H . anomala as an opportunistic yeast pathogen for the first time in Alberta, Canada . Previously published cases of H . anomala infections are reviewed.

Mycopathologia, 1992 Mar, 117(3), 139 - 44
Detection of cellular immunity with the soluble antigen of the fungus Sporothrix schenckii in the systemic form of the disease; Carlos IZ et al.; Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions . The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses . In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S . schenckii . The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks . The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals) . Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen . This depression frequently indicates worsening of the disease, with greater involvement of the host . This is a promising field of research for a better understanding of the pathogeny of this mycosis.

Development, 1992 Mar, 114(3), 681 - 7
Inducible cell ablation in Drosophila by cold-sensitive ricin A chain; Moffat KG et al.; We have developed a system for temperature-inducible killing of specific cells in the fruitfly Drosophila melanogaster . The system overcomes many of the limitations of existing cell ablation methods and is in principle applicable to any non-homeothermic eukaryote . Temperature-sensitive and cold-sensitive mutations in the ricin toxin A chain (RTA) of castor bean were generated in yeast . One cold-sensitive mutation, RAcs2, produced temperature-dependent ablation of eye cells in Drosophila when expressed under control of the eye-specific sev enhancer . At 29 degrees C, cell death was observed within 7 hours in the developing eye and no obvious toxic effects were observed elsewhere; at 18 degrees C, extremely low toxicity was observed . DNA sequencing of RAcs2 revealed a single amino acid substitution in the RTA active site cleft.

J Biochem (Tokyo), 1992 Mar, 111(3), 296 - 301
Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites; Hatsuzawa K et al.; We have recently shown that furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway . In this study, we analyzed molecular and enzymatic properties of furin expressed in Chinese hamster ovary cells using gene transfer techniques . COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region containing the Cys-rich domain and the transmembrane domain is dispensable . A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form . The sequence requirements for precursor cleavage of this truncated furin determined in vitro were similar to those of wild-type furin determined by expression studies in cultured cells . It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency . These observations support the notion that furin is the endogenous endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites.

Bioessays, 1992 Mar, 14(3), 151 - 60
Sorting of proteins to the vacuoles of plant cells; Vitale A et al.; The secretory system of plant cells sorts a large number of soluble proteins that either are secreted or accumulate in vacuoles . Secretion is a bulk-flow process that requires no information beyond the presence of a signal peptide necessary to enter the endoplasmic reticulum . Many vacuolar proteins are glycoproteins and the glycans are often modified as the proteins pass through the Golgi complex . Vacuolar targeting information is not contained in glycans as it is in animal cells; rather, targeting information is in polypeptide domains as it is in yeast cells . Several such domains have now been identified, but these show little or no amino acid sequence homology . We discuss the possibilities that targeting of protein to plant vacuoles may involve receptors as well as aggregation of protein at low pH.

Plant Mol Biol, 1992 Mar, 18(5), 909 - 19
Cytoplasmic ribosomal protein S15a from Brassica napus: molecular cloning and developmental expression in mitotically active tissues; Bonham-Smith PC et al.; We have isolated two cDNA clones which appear to encode the 40S ribosomal subunit protein S15a from Brassica napus (oilseed rape) . The open reading frame in both clones contains 390 bases, encoding a deduced polypeptide sequence of 130 amino acids (100% homology between clones) with 76% sequence identity to the N-terminal 37 amino acids of the rat ribosomal protein S15a and 80% identity to the S24 polypeptide of yeast . Both the yeast and rapeseed proteins have a net positive charge of +9 and the rapeseed S15a protein has a molecular mass of 14778 Da compared to 14762 Da for the yeast protein . The rapeseed ribosomal protein S15a is encoded by a small multi-gene family with at least two actively transcribed members . A single transcript of ca . 1.0 kb, corresponding to ribosomal protein S15a, is abundant in actively dividing tissues such as apical meristem, flower buds and young leaves and less abundant in mature stem and fully expanded leaves.

New Biol, 1992 Mar, 4(3), 173 - 87
Proteasomes: protein and gene structures; Tanaka K et al.; Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides . These particles have a latent multicatalytic proteinase activity . Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin . This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells . We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution . Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene . Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell . In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes.

Clin Infect Dis, 1992 Mar, 14 Suppl 1, S30 - 6
Virulence properties and nonimmune pathogenetic mechanisms of fungi; Vartivarian SE; This article summarizes some of the potential fungal virulence factors, their effect on the host's defense systems, and their regulation by host factors . Immunopathogenesis is not discussed, but the role of adherence by fungal organisms to human surfaces and foreign bodies in pathogenesis is described . Dimorphism and, less commonly, phenotypic switching may play important roles in initiating and establishing infections by several fungi . Toxins, especially exotoxins, do not seem to participate significantly in pathogenesis; however, various enzymes (proteases, phospholipases) may represent virulence properties of Candida, Aspergillus, and a number of other fungi . The interaction of the organisms with their hormonal milieu, the iron-scavenging capacities of various fungi, and their potential role in pathogenesis are delineated . The immunosuppressive effects of certain fungal antigens, such as yeast mannans, are discussed.

Clin Infect Dis, 1992 Mar, 14 Suppl 1, S148 - 53
Pathogenesis and treatment of recurrent vulvovaginal candidiasis; Sobel JD; In contrast to women who experience infrequent episodes of candidal vaginitis, patients with chronic and recurrent candidal vaginitis rarely have recognizable precipitating or causal factors . Analysis of vaginal yeast isolated from women with recurrent candidal vaginitis uncommonly reveals a higher percentage of non-albicans Candida species . There is no indication that resistance to azoles is a causal factor, and no other fungal virulence factors have been identified to explain the repeated attacks . Strain typing of sequential clinical isolates by means of molecular techniques indicate a pattern of relapse due to persistent yeast in the vagina rather than frequent vaginal reinfection . Attempts to reduce the number of attacks by treating sexual partners and suppressing a gastrointestinal tract focus have failed . Recent immunological studies suggest the possibility that an acquired Candida antigen-specific immunological deficiency results in uncontrolled vaginal Candida proliferation and hence repeated clinically evident attacks . Although no definitive cure for recurrent candidal vaginitis exists, numerous therapeutic maintenance regimens with azoles are available that effectively control symptomatic infection.

Mol Biol Evol, 1992 Mar, 9(2), 278 - 84
Two ascomycete classes based on fruiting-body characters and ribosomal DNA sequence; Berbee ML et al.; Traditional fruiting body-based classification of ascomycetes has been under attack for 2 decades . Fruiting-body types can converge, and few researchers now assume that either the closed fruiting bodies (cleistothecia) characterizing the class Plectomycetes or the flask-shaped fruiting bodies (perithecia) characterizing the class Pyrenomycetes are stable, unifying characters . Unless we identify characters uniting major ascomycete groups, orders of ascomycetes remain narrowly defined, and supraordinal classification is impossible . We sequenced both strands of 18s rDNA from nine ascomycete fungi, adding three sequences from GenBank into our analysis . The phylogeny, inferred from 162 informative sites in 1,700 bp of DNA sequence data and using yeast as an outgroup, divided the fungi into two groups correlating well both with fruiting-body type and with the traditional classes Plectomycetes and Pyrenomycetes . Each group received strong statistical support . Genera producing cleistothecia, such as Talaromyces (with a Penicillium asexual state) and the human pathogen Ajellomyces capsulatus (causing histoplasmosis), fall within the plectomycete group . Plectomycetes also includes Eremascus albus and the bee pathogen Ascosphaera apis, although both lack typical fruiting bodies . The Dutch elm disease fungus groups with pyrenomycetes such as Neurospora, in spite of its confusing mixture of class-level characters.

Genomics, 1992 Mar, 12(3), 581 - 9
The identification and characterization of KRAB-domain-containing zinc finger proteins; Constantinou-Deltas CD et al.; The zinc finger motif is a highly conserved tandemly repeated sequence of 28-30 amino acids that was first identified in transcription factor TFIIIA from Xenopus laevis . Subsequently, similar motifs were found and characterized in many genes from mammalian genomes and the genomes of lower eukaryotes such as Drosophila and yeast, thereby defining a large superfamily of genes . Non-finger-coding modules conserved among members of subfamilies of zinc finger genes have been described in the murine genome (finger-associated boxes, or FAX domain) and the human genome (Kruppel-associated boxes, or KRAB domain) . Here we report the identification and partial characterization of more members of the human KRAB-containing subfamily of genes . Based on Southern blot hybridization experiments, they also are zinc-finger-coding genes . All members share a highly homologous 42-amino-acid-long A element of the described KRAB domain . The conservation extends to the murine developmentally expressed zinc finger gene, mKr2 . The homologous sequences, however, are part of the 5'-untranslated region . In all cases for which there is adequate information, the KRAB domain is found at the NH2-terminus of the respective protein . In one zinc-finger-encoding cDNA clone that we characterized further in this work, BRc1744 (ZNF45), the KRAB domain most probably constitutes the entire second exon of the gene . Based on the data, it is tempting to speculate that the FAX- and KRAB-containing zinc finger genes define subfamilies of genes with overlapping functions that participate in the regulation of common or similar developmental programs.

Oncogene, 1992 Mar, 7(3), 589 - 96
The Myb DNA-binding domain is highly conserved in Dictyostelium discoideum; Stober-Grasser U et al.; The c-myb proto-oncogene encodes a protein that is highly conserved among birds and mammals . The amino-terminal domain of c-Myb contains three imperfect tandem repeats of approximately 50 amino acids each . This domain is required for DNA binding and has also been conserved to varying degrees in invertebrates, plants and yeast . Given that myb-related genes appear to control cellular differentiation in a variety of eucaryotic systems, the presence of a myb gene in the cellular slime mold Dictyostelium discoideum might provide a tractable system for studying the role of myb in differentiation . Degenerate oligonucleotide primers encoding regions that are highly conserved in the vertebrate and Drosophila Myb DNA-binding domains were used to amplify a related domain from Dictyostelium genomic DNA, which was then used to isolate a genomic clone . The putative DNA-binding domain of Dictyostelium Myb is as closely related to vertebrate c-Myb as is Drosophila Myb (65% identity), whereas the known Myb-related proteins of plants and yeast are more distantly related . The conserved domain of Dictyostelium Myb is capable of binding to the same DNA sequence as the vertebrate and Drosophila Myb proteins . The remainder of the deduced amino acid sequence of Dictyostelium Myb shows no homology to the divergent domains of the known animal, plant and yeast Myb-related proteins . Evolutionary analysis implies that the duplications that generated the repeats of the Myb DNA-binding domain began prior to the divergence of animals, plants, cellular slime molds and yeast.

Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1875 - 9
The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression; Ballard DW et al.; Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-kappa B . Functional analyses of chimeric proteins composed of NF-kappa B p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-kappa B p65 comprise a potent transcriptional activation domain . Transient transfection of human T cells with an expression vector encoding NF-kappa B p65, but not NF-kappa B p50, produced marked transcriptional activation of a basal promoter containing duplicated kappa B enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus . These stimulatory effects of NF-kappa B p65 were synergistically enhanced by coexpression of NF-kappa B p50 but were completely inhibited by coexpression of the v-rel oncogene product . Together, these functional studies demonstrate that NF-kappa B p65 is a transactivating subunit of the heterodimeric NF-kappa B complex and serves as one cellular target for v-Rel-mediated transcriptional repression.

Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1641 - 5
Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini; Beushausen S et al.; Previously, two forms of cAMP-dependent protein kinase catalytic subunit generated by mutually exclusive use of two internal exon cassettes (A1 and A2) were demonstrated in Aplysia neurons . Here, it is shown that there also exist catalytic subunits with alternative N termini derived from two exons, N1 and N2, expressed in combination with either of the internal cassettes . Processed transcripts including N1 or N2 sequences are of about equal abundance in the nervous system, arise through alternative promoter use, and encode catalytically active polypeptides . The N2 amino acid sequence is 21 residues longer than the N1 sequence and is homologous to the nonmyristoylated N terminus of the TPK1 gene product, a yeast catalytic subunit homolog . These data support the view that cAMP-dependent protein kinase activity in Aplysia neurons is produced by a complex array of regulatory and catalytic subunits that generate multiple holoenzymes with a spectrum of properties.

Eur J Biochem, 1992 Mar 1, 204(2), 865 - 73
Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria; Bourguignon J et al.; L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration . We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor . The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues . The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA . Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved . Most of the residues located in or near the active sites remain unchanged . The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes . Confidence in this conclusion comes from the following considerations . First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected . Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues . In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves . Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.

Arch Biochem Biophys, 1992 Mar, 293(2), 382 - 90
Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation; Sun AQ et al.; Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis . We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein . CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI . Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer . Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability . The deamidated enzyme was more susceptible to proteases and denaturing conditions . Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond . The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity . The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation . The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.

Andrologia, 1992 Mar-Apr, 24(2), 87 - 93
Immunosuppression by human seminal plasma fractionated by DEAE Sephadex A-50 ion exchange chromatography; Haq A et al.; We fractionated the whole human seminal plasma on DEAE Sephadex A-50 ion exchange columns . Complete separation was achieved in seven peaks using different salt concentrations in phosphate buffer pH 6 . The seminal plasma proteins were separated by sodium dodecyl-sulphate polyacrylamide gel electrophoresis . Human seminal plasma (SP) and its fractions were used in mixed lymphocyte reaction in vitro . Fractions 3, 4, and 7 were found to suppress the proliferation of human peripheral blood mononuclear cells to phytohemagglutinin and pokweed mitogen at a concentration of 10 micrograms ml-1 while stimulatory effect was observed at lower concentrations (1 microgram and 2.5 micrograms ml-1) . Whole human SP and other fractions failed to suppress the proliferation of lymphocytes in vitro . Furthermore, the effect of human SP and its fractions was also investigated on phagocytic function of polymorphonuclear leukocytes (PMNs) using luminol dependent chemiluminescence assay stimulated with phorbol myristate acetate and opsonized yeast . Fractionated SP was found to have a suppressive effect on the luminol-dependent chemiluminescence of PMNs in the whole blood.

J Am Acad Dermatol, 1992 Mar, 26(3 Pt 2), 452 - 7
A double-blind, placebo-controlled, multicenter trial of lithium succinate ointment in the treatment of seborrheic dermatitis . Efalith Multicenter Trial Group.
{Antifungal varnish in the treatment of denture stomatitis}
Carlino P, Lang R, Budtz-Jorgensen E.

Division de Gerodontologie et prothese adjointe, Universite de Geneve, SuisseThe efficacy of a single-dose application of miconazole varnish in the treatment of denture stomatitis was compared with miconazole gel applied three times/day for 15 days . Among 288 wearers of complete dentures 50 patients severely affected by denture stomatitis and heavily colonized by Candida, were selected . The patients were assigned randomly to two groups: a miconazole varnish group and a gel group . All patients were examined 7 times: before starting the treatment (day 0); during treatment (day 3, 7 and 14); after treatment (day 21, 28 and 35) . At each examination a photograph of the palatal mucosa was obtained and quantitative cultures of Candida from the lesions and the fitting denture surface were performed . At day 14 the inflammation was reduced to the same degree in the two groups of patients . A comparison of the antifungal effect, i.e . reduction of the yeast score, showed no significant difference between the two groups . Maximum reduction of the number of yeasts was obtained at day 14 . From that moment, recolonization started; thus, about 60% of the treated patients whether it was with the gel or the varnish was positive of Candida by day 35 . In conclusion, the present study showed no difference in the clinical and antifungal effect of a single-dose application of miconazole varnish compared with miconazole gel . The varnish seemed to be better with respect to the posology as the total dose of miconazole is minimal and only one application is necessary . Treatment with the miconazole varnish seems particularly indicated in debilitated and non-cooperative patients suffering from Candida-associated denture stomatitis.

Biochem Cell Biol, 1992 Mar-Apr, 70(3-4), 207 - 14
Mitochondrial activity and heat-shock response during morphogenesis in the pathogenic fungus Histoplasma capsulatum; Patriarca EJ et al.; Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms . In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 degrees C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium- to yeast-phase transition . The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature . We have found that by inducing thermotolerance, i.e., an initial incubation at 34 degrees C, the thermosensitive attenuated Downs strain of H . capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures . Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 degrees C proceeds at rates comparable to those of the virulent strains.

Genes Dev, 1992 Mar, 6(3), 345 - 55
Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands; Jhappan C et al.; Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV) . Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6 . To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences . All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed . Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age . Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice . The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated . In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis . These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia.

Mol Phylogenet Evol, 1992 Mar, 1(1), 59 - 71
Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence; Berbee ML et al.; Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release . Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms . The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes . The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently . Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge . In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates . A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test . Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge . Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA . The transition-to-transversion ratio was 1.9 . Of all 12 possible substitutions, 29% were from U to C . At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4) . In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5).

Eur J Biochem, 1992 Mar 1, 204(2), 841 - 6
Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria; Jordi W et al.; In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics . In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein {Rietveld, A . & de Kruijff, B . (1984) J . Biol . Chem . 259, 6704-6707; Dumont, M . E . & Richards, F . M . (1984) J . Biol . Chem . 259, 4147-4156} . Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria . In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer . The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins . The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.

Nucleic Acids Res, 1992 Feb 25, 20(4), 735 - 45
Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase delta; Yang CL et al.; The cDNA of human DNA polymerase delta was cloned . The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa . Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb . Monoclonal and polyclonal antibodies to the C-terminal 20 residues specifically immunoblotted the human pol delta catalytic polypeptide . A multiple sequence alignment was constructed . This showed that human pol delta is closely related to yeast pol delta and the herpes virus DNA polymerases . The levels of pol delta message were found to be induced concomitantly with DNA pol delta activity and DNA synthesis in serum restimulated proliferating IMR90 cultured cells . The human pol delta gene was localized to chromosome 19 by Southern blotting of EcoRI digested DNA from a panel of rodent/human cell hybrids.

J Biol Chem, 1992 Feb 25, 267(6), 3589 - 96
The effects of dNTP pool imbalances on frameshift fidelity during DNA replication; Bebenek K et al.; The use of unequal concentrations of the four deoxynucleoside triphosphates (dNTPs) in DNA polymerization reactions alters base substitution error rates in a predictable way . Less is known about the effects of substrate imbalances on base addition and deletion error rates . Thus, we examined pool bias effects on frameshift fidelity during DNA synthesis catalyzed by replicative DNA polymerases . Imbalanced pools altered the frameshift fidelity of the human immunodeficiency virus type-1 reverse transcriptase . Both mutagenic and antimutagenic effects were observed for minus-one, plus-one, and minus-two nucleotide errors, in a highly sequence-specific manner . Most of this specificity can be rationalized by either of two models . One involves frameshifts initiated by pool bias-induced nucleotide misinsertion, and the other involves pool bias-initiated template-primer slippage . Several examples of complex mutations were also recovered more than once in small mutant collections . These contained closely spaced single-base substitution and minus-one base frameshift changes . The two changes occurred at a frequency much higher than predicted if they were generated independently . This suggests that when the polymerase makes one mistake, the probability that it will make a second mistake within the next few incorporations increases significantly . Perturbation of dNTP pools also affected the frameshift fidelity of the replicative yeast DNA polymerase alpha . In reactions containing a low concentration of one dNTP, the error rate increased for one-nucleotide deletions at homopolymeric template nucleotides complementary to the dNTP whose concentration was low . We extended this approach to determine the frameshift fidelity of simian virus 40 origin-dependent semiconservative replication of double-stranded DNA in extracts of human cells . In reactions performed with an equal concentration of all four dNTPs, replication was highly accurate for minus-one-nucleotide errors . However, when the concentration of one dNTP was decreased, the replication error rate increased at complementary, homopolymeric template positions . This response provides an approach for describing frameshift accuracy during replication of the leading and lagging strands.

J Biol Chem, 1992 Feb 25, 267(6), 3577 - 80
Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase; Wang K et al.; Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site . In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells . The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis . The binding of {3H}ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi . The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM . The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480 . The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM) . These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate . It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.

Nature, 1992 Feb 20, 355(6362), 733 - 5
Dynamin is a GTPase stimulated to high levels of activity by microtubules; Shpetner HS et al.; Dynamin was initially identified in calf brain tissue as a protein of relative molecular mass 100,000 which induced nucleotide-sensitive bundling of microtubules . Purified dynamin showed only trace ATPase activity . But in combination with an activating factor removed during the purification, it exhibited microtubule-activated ATPase activity and dynamin-induced bundles showed evidence of ATP-dependent force production . Dynamin is the product of the Drosophila gene shibire, which has been implicated in synaptic vesicle recycling and, more generally, in the budding of endocytic vesicles from the plasma membrane . Dynamin also shows extensive homology with proteins that participate in vacuolar protein sorting and spindle pole-body separation in yeast, and in interferon-induced viral resistance in mammals . All members of this family contain consensus sequence elements consistent with GTP binding near their amino termini, although none has been shown to have GTPase activity . We report here that dynamin is a specific GTPase which can be stimulated to very high levels of activity by microtubules.

Eur J Biochem, 1992 Feb 15, 204(1), 21 - 30
Cross-species comparison of the sequence of the leukaemia inhibitory factor gene and its protein; Willson TA et al.; Leukaemia inhibitory factor (LIF) is a pleiotropic growth factor active in diverse cell systems in both the adult and the embryo . The LIF gene from a number of mammalian species is highly conserved . The ovine and porcine LIF genes were cloned, sequenced and compared to the previously published murine and human LIF gene sequences . While the coding regions of the LIF gene are highly conserved, the non-coding regions are largely non-conserved . In a region of approximately 340 bp, the 5' end of the translational initiation codon is highly conserved (84%) . This region includes four conserved TATA boxes, two transcriptional start-sites identified in the murine gene and the minimal region required to function as the LIF promoter . A sequence in the murine gene adjacent to this highly conserved region which appears to contain a negative control element is, however, poorly conserved between the four species compared, except for a sequence of 16 conserved nucleotides . Within the largely non-conserved first intron, there is a block of approximately 150 nucleotides which is highly conserved between all four species (approximately 72%) . However, a sequence in intron 1 of the murine LIF gene which corresponds to an alternative exon of a putative variant LIF transcript is very poorly conserved between species, with only relics of this exon evident in the other three species . A comparison of the five LIF protein sequences available (murine, rat, human, ovine and porcine) revealed that the protein displays a high degree of similarity, ranging from 74% between mouse and sheep to 92% between rat and mouse . Several large blocks of absolutely conserved amino acid sequence were identified . The ovine LIF gene was modified to allow production of recombinant ovine LIF in yeast cells, which was shown to be biologically active on murine cells.

J Biol Chem, 1992 Feb 15, 267(5), 2841 - 4
Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration; Lu XP et al.; Cyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J . L . (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp . 323-328, Raven Press, New York), yeast cell cycles (Nurse, P . (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R . L., Marshak, R . R., and Roberts, J . M . (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T . (1991) Nature 350, 462-463) . The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown . Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats . To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules . The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively . These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.

Arch Biochem Biophys, 1992 Feb 14, 293(1), 158 - 66
Identification, purification, and characterization of S-adenosyl-L-methionine: isoliquiritigenin 2'-O-methyltransferase from alfalfa (Medicago sativa L.); Maxwell CA et al.; An O-methyltransferase (OMT) which methylates the 2'-hydroxyl of isoliquiritigenin (2',4,4'-trihydroxychalcone) was identified in alfalfa (Medicago sativa L.) seedlings and cell cultures . The OMT activity increased during early stages of seedling development and was predominantly located in roots . Treatment of alfalfa cell cultures with an elicitor from yeast resulted in a fivefold increase in chalcone OMT activity, whereas treatment of seedlings with CuCl2 caused a reduction in activity . The chalcone OMT was purified to near homogeneity from elicited alfalfa cell cultures . Only one form of the enzyme was found . It consisted of an active monomer of subunit Mr 43,000 which could be photoaffinity labeled with S-adenosyl-L-{methyl-3H}methionine . The purified OMT had a pH optimum of 9.0, pI of 4.7, and was highly specific for the 2'-hydroxyl of 2',4,4'-trihydroxychalcone, with essentially no activity toward narigenin chalcone, caffeic acid, or daidzein . Kinetic analysis indicated a sequential bi bi mechanism with Km values of 2.2 and 17.7 microM for 2',4,4'-trihydroxychalcone and S-adenosyl-L-methionine, respectively . S-Adenosyl-L-homocysteine was a potent inhibitor . The chalcone OMT represents the third distinct OMT isolated from alfalfa cell cultures.

J Immunol Methods, 1992 Feb 14, 147(1), 125 - 34
Caveats for the use of surface-adsorbed protein antigen to test the specificity of antibodies; Schwab C et al.; Rabbit antisera against apo-cytochrome c, which was prepared by removal of the covalently bound heme prosthetic group from yeast iso-1 cytochrome c, were tested for reactivity against native yeast iso-1-cytochrome c . When the antigen was adsorbed to a microtiter plate in a conventional enzyme-linked immunosorbent assay (ELISA), the antisera were unable to distinguish between their cognate antigen apo-cytochrome c, a random coil protein, and native cytochrome c, a small globular protein of remarkable conformational stability in solution . However, when the assay was conducted under conditions where antigen and antibody were free to associate in solution, that is in a solution-phase radioimmunoassay (RIA), the antisera were highly specific for apo-cytochrome c . Similarly, antibodies induced by native cytochrome c and discriminating strongly between native and apo-cytochrome c in a solution-phase RIA, did not distinguish between native and apo-cytochrome c in a solid-phase ELISA . This discrepancy of results obtained by different immuno assay procedures clearly indicates that adsorption to plastic alters the antigenic structure of even a conformationally stable protein such as cytochrome c . A conventional solid-phase ELISA strongly selects for those antibodies that recognize the unfolded antigen . The results presented warrant serious thoughts about previous reports on anti-peptide antibodies reacting with native whole protein molecules, as tested by those ELISA procedures that have the protein antigen adsorbed to plastic.

Nucleic Acids Res, 1992 Feb 11, 20(3), 475 - 8
Recognition nucleotides for human phenylalanyl-tRNA synthetase; Nazarenko IA et al.; The specificity of the interaction between tRNAPhe and phenylalanyl-tRNA synthetase isolated from human placenta was investigated . Using yeast tRNAPhe transcripts with different point mutations it was shown that all the five recognition points for the yeast phenylalanyl-tRNA synthetase (G20, G34, A35, A36 and A73) are also important for the reaction catalyzed by the human enzyme . A set of mutations in nucleotides involved in tertiary interactions of tRNAPhe revealed that mutations which maintained the proper folding of the molecule had almost no influence on the efficiency of aminoacylation . The most striking difference between the yeast and human phenylalanyl-tRNA synthetases involved a mutation in the lower two base pairs of the anticodon stem . This mutation did not affect aminoacylation with the yeast enzyme, but greatly reduced activity with human phenylalanyl-tRNA synthetase.

Nature, 1992 Feb 6, 355(6360), 548 - 51
Cloning of the essential myotonic dystrophy region and mapping of the putative defect; Aslanidis C et al.; Myotonic dystrophy is a common dominant disorder (global incidence of 1:8,000) with variable onset and a protean nature of symptoms mainly involving progressive muscle wasting, myotonia and cataracts . To define the molecular defect, we have cloned the essential region of chromosome 19q13.3, including proximal and distal markers in a 700-kilobase contig formed by overlapping cosmids and yeast artificial chromosomes (YACs) . The central part of the contig bridges an area of about 350 kilobases between two new flanking crossover borders . This segment has been extensively characterized through the isolation of five YAC clones and the subsequent subcloning in cosmids from which a detailed EcoRI, HindIII, MluI and NotI restriction map has been derived . Two genomic probes and two homologous complementary DNA probes were isolated using the cosmids . These probes are all situated within approximately 10 kilobases of genomic DNA and detect an unstable genomic segment in myotonic dystrophy patients . The length variation in this segment shows similarities to the instability seen at the fragile X locus . The physical map location and the genetic characteristics of the length polymorphism is compatible with a direct role in the pathogenesis of myotonic dystrophy.

J Biol Chem, 1992 Feb 5, 267(4), 2200 - 8
cDNA cloning for mouse thymocyte-activating molecule . A multifunctional ecto-dipeptidyl peptidase IV (CD26) included in a subgroup of serine proteases; Marguet D et al.; Thymocyte-activating molecule (THAM) was initially characterized as a developmentally regulated, dimeric cell-surface molecule capable of activating mouse thymocytes and T lymphocytes upon monoclonal antibody (mAb)-mediated cross-linking . We recently obtained structural evidence indicating that this molecule is the mouse homologue of the human T cell-activating ectoenzyme CD26 (dipeptidyl peptidase IV, DPP IV) . We describe here the cloning and the characterization of THAM cDNA . Two clones (3.3 and 2.8 kilobases) were isolated . THAM-3.3 cDNA contains an open reading frame of 2,280 nucleotides that encodes a protein of 760 amino acids having a calculated size of 87,500 Da . Complete N-glycosylation at each of the nine potential sites would result in a mature 110,000-Da molecule . Protein sequence comparisons revealed a significant homology (in particular in the COOH-terminal domain) between THAM and the rat or human DPP IV or the yeast dipeptidyl aminopeptidase B molecules (92, 85, and 30% sequence identity, respectively) . Structural comparison of serine proteases (i.e . acyl-amino acid hydrolase or prolyl endopeptidase) with the most conserved domain of THAM identified a stretch of 200 amino acids containing a putative catalytic triad arranged in a novel topological order (Ser-624, Asp-702, and His-734) thereby defining a subfamily of nonclassical serine proteases . Expression of THAM during thymus ontogeny was found to be mainly regulated at the transcriptional level as determined by RNase protection assay . To investigate directly some of the functions which have been ascribed to DPP IV, we transfected an ovalbumin/Aq-reactive, THAM- T hybridoma cell line with THAM-3.3 cDNA . The resultant transfectants acquired (i) DPP IV enzymatic activity that precisely paralleled the density of surface-expressed THAM; (ii) an Mr = 115,000 (reduced) and 110,000/128,000 (nonreduced) molecule that could be immunoprecipitated by the THAM-specific mAb H194-112; and (iii) the capacity of being triggered by this mAb to release interleukin-2 . These data indicate that a single cDNA species can encode a multifunctional molecule (e.g . activation signal-transducing structure and ectopeptidase), the heterodimeric state of which very likely results from a differential post-translational modification of the same protein core.

J Biol Chem, 1992 Feb 5, 267(4), 2294 - 302
Cloning, structural analysis, and expression of the glycogen phosphorylase-2 gene in Dictyostelium; Rutherford CL et al.; The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity . The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits . The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography . The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template . The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene . These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene . The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively . The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4 . An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region . The CAA repeat was transcribed, as shown by the sequence of cDNA . Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively . Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development . Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development . However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide . Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.

Carcinogenesis, 1992 Feb, 13(2), 217 - 22
Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4'-methylene-bis(2-chloroaniline); Yun CH et al.; 4,4'-Methylene-bis(2-chloroaniline) (MOCA) can produce tumors in rodents and dogs and an increased incidence of bladder tumors has been reported in exposed workers . It is therefore of interest to identify the human cytochrome P450 (P450) enzymes involved in MOCA N-oxidation, the primary reaction involved in the formation of an electrophilic product . Human liver microsomes were fractionated and MOCA N-oxidation activity was monitored through the procedure . The most active enzyme fraction corresponded to P450 3A4, as determined by immunochemical assays and N-terminal amino acid sequence analysis . Yeast recombinant P450 3A4 also had MOCA N-oxidation activity . Purified human liver P450 2A6 showed catalytic activity; however, anti-P450 2A6 inhibited less than 20% of the microsomal activity while anti-P450 3A4 inhibited up to 75% . Levels of marker activities of both P450 3A4 (nifedipine oxidation) and P450 2A6 (coumarin 7-hydroxylation) were measured in a set of human liver microsomes and both were correlated with MOCA N-oxidation rates . Gestodene and troleandomycin inhibited up to half of the microsomal MOCA N-hydroxylation activity but 7,8-benzoflavone showed only slight inhibition . Anti-P450 3A4 inhibited (up to 80% of) the microsomal transformation of MOCA to a product genotoxic as judged by bacterial SOS response . The work indicates that P450 3A4 makes a major contribution to human liver microsomal MOCA N-oxidation, and P450 2A6 has a minor role . P450 1A2, which catalyzes the hydroxylation of many arylamines, does not contribute to a great extent.

Urol Clin North Am, 1992 Feb, 19(1), 143 - 7
Balanitis and balanoposthitis; Vohra S et al.; Balanitis is an inflammation of the glans penis . There are several etiologic agents, including bacterial and yeast infections, parasitic infestations, and trauma or irritants . Plasma-cell balanitis and balanitis xerotica obliterans are two distinct clinical entities . The authors review the clinical and pathologic features and the treatment options for these conditions.

Eur J Biochem, 1992 Feb 1, 203(3), 459 - 66
Engineering mammalian aspartyl-tRNA synthetase to probe structural features mediating its association with the multisynthetase complex; Mirande M et al.; Aspartyl-tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl-tRNA synthetases . The cDNA encoding cytoplasmic rat liver aspartyl-tRNA synthetase was previously cloned and sequenced . This work reports the identification of structural features responsible for its association within the multisynthetase complex . Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed . A wild-type rat liver aspartyl-tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino-terminal extremity does not . A chimeric enzyme, made of the amino-terminal moiety of rat liver aspartyl-tRNA synthetase fused to the catalytic domain of yeast lysyl-tRNA synthetase, has been expressed in Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase . The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells . These results establish that the 3.7-kDa amino-terminal moiety of mammalian aspartyl-tRNA synthetase mediates its association with the other components of the complex . In addition, the finding that yeast lysyl-tRNA synthetase cannot replace the aspartyl-tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well-defined structural organization.

Mol Cell Biol, 1992 Feb, 12(2), 865 - 75
Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases; Oommen A et al.; tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions . Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini . Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus . Sequences downstream of nucleotide 74, however, are always removed . Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions . The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all . A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH . Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74 . In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH) . This specificity is consistent with that of all previously characterized nuclear enzyme preparations . We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76 . This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

Blood, 1992 Feb 1, 79(3), 610 - 8
A-T-rich scaffold attachment regions flank the hematopoietic serine protease genes clustered on chromosome 14q11.2; Hanson RD et al.; We have analyzed approximately 70 kb of the chromosome 14q11.2 hematopoietic serine protease gene cluster for the presence of nuclear scaffold attachment regions (SARs) . At least 12 potential attachment sites were identified . SARs are present on both sides of the CGL-1/CSP-B and CGL-2/CCP-X genes and upstream from the cathepsin G (CG) gene . We have further characterized the SARs immediately flanking the cytotoxic lymphocyte-specific CGL-1/CSP-B gene . These 5' and 3' SARs are highly A-T-rich, contain multiple attachment sites, and are associated with the scaffolds of nuclei derived from both lymphoid and erythroid cell lines . These SARs contain multiple consensus elements frequently associated with A-T-rich sequences, including the vertebrate topoisomerase II (topo II) consensus sequence, the A-box and T-box elements, and the yeast autonomous replicating sequence (ARS) . The potential role for the nuclear scaffold in the transcriptional regulation of CGL-1/CSP-B expression is discussed.

Curr Opin Genet Dev, 1992 Feb, 2(1), 45 - 52
Learning about cancer genes through invertebrate genetics; Hoffmann FM et al.; Genetic studies in yeast, nematodes and Drosophila are revealing the signal transduction pathways that regulate differentiation and cell proliferation . Some of the critical molecules involved are homologous to proto-oncogenes and others are likely to be analogous to the products of tumor suppressor genes.

J Cell Biochem, 1992 Feb, 48(2), 129 - 35
Identification of a core motif that is recognized by three members of the HMG class of transcriptional regulators: IRE-ABP, SRY, and TCF-1 alpha; Alexander-Bridges M et al.; Insulin induces glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene transcription in part by regulating one or more proteins that bind a cis-acting element, IRE-A . We have recently cloned a protein, IRE-ABP, that binds the IRE-A element . IRE-ABP is a member of the HMG class of transcriptional regulators and is 67% identical within its HMG box domain to the candidate gene for the testis-determining factor, SRY . IRE-ABP and SRY share binding specificity for the IRE-A motif . This sequence is also highly conserved with a core motif, 5'-Py-ctttg(a/t)-3', contained in T-cell specific genes that have high affinity for TCF-1 alpha, another member of the HMG class of transcriptional regulators . Thus, diverse members of the HMG family interact with similar nucleotide sequences to regulate expression of genes that initiate and maintain the differentiated phenotype . We have found this core motif in the upstream region of many genes that are positively and negatively regulated by insulin . These observations suggest that IRE-ABP or a related family member may coordinate the expression of these genes . The HMG family of proteins has diverse functions ranging from the regulation of differentiation and mating type in yeast to the regulation of tissue- and species-specific gene expression in mammals . Insulin regulates GAPDH gene transcription in a tissue-specific manner . We propose that members of the IRE-ABP family play an important role in controlling tissue specificity of the insulin response.

Sci China B, 1992 Feb, 35(2), 155 - 61
Structure of the mitochondrial URFA6L gene and tRNA(Lys) gene from CARP; Wang GF et al.; The carp mitochondrial URFA6L gene consists of 165 base pairs . The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene . Their nucleotide sequences exhibit 68% homology . The carp URFA6L gene encodes a protein of 54 amino acids . The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids (proline, tryptophan, leucine, isoleucine and tyrosine) . A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase 8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function as ATPase8 . The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in clover-leaf secondary structure . Similar to amphibian and mammalian mitochondrial tRNA(Lys) genes, the carp mitochondrial tRNA(Lys) gene also has some unusual structural features as compared with its cytoplasmic counterpart . A comparison between the nucleotide sequences of the tRNA(Lys) gene from 7 vertebrates showed that the most conservative portions are the anticodon loop, nucleotides 8 and 9, the variable loop, the anticodon stem and the aminoacyl stem . The least conservative portions are the D-loop and the T-loop . These structural features may show that the mitochondrial tRNA(Lys) has a different interaction with mitochondrial ribosome.

Eur J Immunogenet, 1992 Feb-Apr, 19(1-2), 45 - 55
Complexity in the major histocompatibility complex; Trowsdale J et al.; The human major histocompatibility complex (MHC) is one of the most intensively studied regions of the human genome, containing over 70 known genes and spanning about 4 million base pairs (4 Mbp) of DNA on chromosome 6p21.3 (Klein, 1986) . It can be divided up into three regions: the class I region (telomeric), the class II region (centromeric), and the class III region (between class I and II), which includes the complement component genes C2, C4, and Bf (Trowsdale & Campbell, 1988) . The MHC has been mapped in detail using pulse field gel electrophoresis (PFGE) and by cloning in yeast artificial chromosome (YAC) and cosmid vectors, revealing long stretches of DNA between the regions as well as between individual class I and class II genes . Novel genes, that have no sequence relationships with class I, class II or complement components, have recently been found in these areas, and we will present an update on these after reviewing the more established loci.

Trends Genet, 1992 Feb, 8(2), 70 - 5
Towards a Drosophila genome map; Hartl DL et al.; A physical map of the genome of Drosophila melanogaster has been created using 965 yeast artificial chromosome (YAC) clones assigned to locations in the cytogenetic map by in situ hybridization with the polytene salivary gland chromosomes . Clones with insert sizes averaging about 200 kb, totaling 1.7 genome equivalents, have been mapped . More than 80% of the euchromatic genome is included in the mapped clones, and 75% of the euchromatic genome is included in 161 cytological contigs ranging in size up to 2.5 Mb (average size 510 kb) . On the other hand, YAC coverage of the one-third of the genome constituting the heterochromatin is incomplete, and clones containing long tracts of highly repetitive simple satellite DNA sequences have not been recovered.

J Gen Microbiol, 1992 Feb, 138 ( Pt 2), 395 - 9
Small subunit ribosomal RNA of Blastomyces dermatitidis: sequence and phylogenetic analysis; Geber A et al.; We determined the small subunit (18S) ribosomal RNA sequence of the dimorphic fungus Blastomyces dermatitidis . The sequence was compared to that of fourteen other eukaryotic organisms, ten of which were higher fungi, and an evolutionary tree was constructed based on these sequences . B . dermatitidis aligned most closely with the Ascomycetes Neurospora crassa and Podospora anserina, in agreement with previous phylogenetic analysis based on morphological criteria . Phase-specific cDNA clones derived by reverse transcription of RNA isolated from the yeast and mycelial phases of B . dermatitidis were also sequenced . The 18S ribosome sequence was found to be the same in both phases . Heterogeneity was found at both the genomic and RNA level at position 1352.

Plant Mol Biol, 1992 Feb, 18(4), 691 - 701
Cloning and sequencing of a cDNA encoding ascorbate peroxidase from Arabidopsis thaliana; Kubo A et al.; A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage lambda gt11 library of cDNA from Arabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced . The cDNA insert hybridized to a 1.1 kb poly(A)+ RNA from leaves of A . thaliana . Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene . The N-terminal amino acid sequence of Arabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach . The predicted amino acid sequence of the mature AP of A . thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochrome c peroxidase of yeast, but less homologous with other plant peroxidases . Amino acid residues at the active site of yeast cytochrome c peroxidase are conserved in the amino acid sequence of Arabidopsis AP . The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3' untranslated region of the cDNA.

Curr Opin Cell Biol, 1992 Feb, 4(1), 80 - 5
Replication of basal bodies and centrioles; Johnson KA et al.; During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle . Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules . Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.

J Anim Sci, 1992 Feb, 70(2), 559 - 65
Supplemental chromium for stressed and growing feeder calves; Chang X et al.; The effects of supplemental chromium (Cr) from high-Cr yeast were investigated with steer calves fed corn silage diets . One hundred eight Charolais-crossed calves, weighing 245 kg after marketing and transport, were allotted to one of four treatments during the initial 28-d stress period: control, .4 ppm of Cr in the diet, long-acting injectable oxytetracycline (LAOTC), and Cr + LAOTC . Those fed Cr received 4 mg of Cr/d for the first 3 d sprinkled onto a small amount of hay over the silage . Chromium without LAOTC increased (P less than .05) ADG by 30% (.61 vs .79 kg/d) and ADG/DMI by 27% (.123 vs .156) . Oxytetracycline alone increased (P less than .05) ADG by 30% and DMI by 15% . Chromium had no effect on morbidity . However, LAOTC tended (P less than .14) to reduce morbidity (26.0 vs 14.0%) after its administration . After d 28, steers were processed . Two weeks later, they were rerandomized within Cr groups to urea-corn vs soybean meal supplementation of corn silage during a 70-d growing period . Level of Cr was reduced to .2 ppm . Jugular blood was collected from eight steers on each treatment on two occasions . Chromium had no effect on ADG or ADG/DMI . However, Cr decreased (P less than .05) serum cortisol (75.0 vs 55.6 nmol/L) . Furthermore, Cr increased (P less than .05) serum immunoglobulin M and total immunoglobulins in calves fed diets with soybean meal but had no effect in calves with urea-corn supplementation.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Biol Interact, 1992 Feb, 81(3), 291 - 306
Studies on Se incorporation in selenoproteins; effects of peroxisome proliferators and hydrogen peroxide generating system; Garberg P et al.; The objective of this study was to characterize the influence of peroxisome proliferation on the metabolism of physiological concentrations of Se . In an initial series of experiments hepatocytes in primary cultures and isolated from ordinary-fed rats, were used . The cells were exposed to 75Se-selenite (30 nM) and after 24 h the labelling of selenoproteins was analysed with SDS-PAGE . Treatments with mono(2-ethylhexyl)phthalate (MEHP; a metabolite of di(2-ethylhexyl)phthalate (DEHP)), nafenopin, decreased oxygen tension and a H2O2 generating system decreased the labelling of a 23-kDa and a 15-kDa protein . The decreased labelling of the 23- and the 15-kDa proteins was usually accompanied by an increased labelling of a 58-kDa protein . Increased oxygen tension induced uncertain effects, possibly due to toxicity . In order to further evaluate the validity of the model, the labelling was also studied in hepatocytes isolated from Se-deficient and torula yeast-fed rats . In these cells there was a decreased labelling of the 23-kDa protein as compared to cells from Se-supplemented controls when 100 nM selenite was used . In in vivo experiments it was found that a DEHP-induced decrease in glutathione peroxidase (GSH-Px) activity was potentiated by high doses of selenite . To a large extent, the labelling data are compatible with enzyme activity data and in vivo data . For example, the decreased labelling of the 23-kDa protein may reflect the decreased GSH-Px activity . It is concluded that the effects induced by MEHP on Se-labelling can be explained by an increase in the steady state level of H2O2.

EMBO J, 1992 Feb, 11(2), 489 - 96
Molecular cloning and characterization of an interferon induced human cDNA with sequence homology to a mammalian peptide chain release factor; Buwitt U et al.; Here we report the molecular cloning of several related human cDNAs from which a full-length sequence can be determined . The cDNAs encode a 2.8 kb mRNA that is strongly induced by interferon (IFN) gamma and the expression of which is not cell-restricted but observed in fibroblasts, macrophages and epithelial cells . The deduced amino acid sequence predicts a protein of 471 amino acids with high sequence similarity to a previously identified rabbit peptide chain release factor . Functional studies to demonstrate release factor activity showed that the protein encoded by this cDNA inhibited the readthrough activity of a yeast UGA suppressor tRNA in an in vitro translation system . The identification of this novel cDNA implies that translational control by IFN induced proteins may not be restricted to the initial steps of protein synthesis but may also act by regulation of peptide chain termination.

Mol Cell Biol, 1992 Feb, 12(2), 784 - 90
Interaction of Wnt-1 proteins with the binding protein BiP; Kitajewski J et al.; The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development . We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters . A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins . We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP . Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP . We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells . The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.

Antibiot Khimioter, 1992 Feb, 37(2), 6 - 11
{Characteristics of obtaining protoplasts from 2 strains of Tolypocladium inflatum subsp . Blastosporum}; Sotnikova IV et al.; The optimal conditions for preparing protoplasts with high yields by using the cells of two (low and high potent) isogenic cyclosporine-producing Tolypocladium strains were developed . A specific medium containing 0.5 per cent yeast autolysate (by dry weight) and 3 per cent glucose was used . When grown on this medium the cells of the highly potent strain 847 acquired a yeast-like shape . High yields of protoplasts prepared from the low potent strain 43 mycelium were obtained via prior incubation with 0.01 M dithiothreitol followed by treatment with a complex of enzymes from Helix pomatia for 1.5 to 2 hours was used . For preparation of the protoplasts with employing the highly potent strain 847 cells the prior incubation with dithiothreitol was not required, but it was necessary to employ a mixture of the enzyme complex (Helix pomatia), drizilase (Irpex lacteus) and chitinase (Streptomyces griseus) for 18 hours . The electron microscopic data on the two isogenic strains and their protoplasts are presented . The protoplasts proved to be a suitable initial material for investigating bioenergetic processes at the subcellular level and further genetic improvement of the strains.

Cell Growth Differ, 1992 Feb, 3(2), 135 - 42
Erks: their fifteen minutes has arrived; Crews CM et al.; In conclusion, a multigene family (ERK) encoding protein kinases that have the capacity to convert tyrosine kinase signals to serine/threonine phosphorylation signals has been identified in animal and yeast cells . Protein kinases from this family have been shown to be phosphorylated on tyrosine and threonine in response to mitogens, as well as to have the capacity to autophosphorylate on these amino acid residues . In contrast, they apparently phosphorylate exogenous substrates on serine and/or threonine . Studies with cultured cells, Xenopus, and sea star oocytes have furthered our understanding of possible functions of Erks in vivo . These enzymes respond immediately to extracellular signals and are involved in G0-G1 transition (cultured cells), as well as in the M phase of oocyte maturation (Xenopus and sea star oocytes) . Their usage of MAPs as substrates in vivo suggests a possible role of Erks in microtubule reorganization . ERK-encoded protein kinases use c-Jun, EGF receptor, and Raf-1 as potential substrates and can also reactivate dephosphorylated S6 kinase in vitro . Taken together, these data suggest that these enzymes play an important role in relaying the mitogenic signal by phosphorylating down-stream kinases and specific transcriptional factors, as well as having possible feedback function in the process of signal transduction . The results from the study of the yeast enzymes are pertinent to Erk activation in cells with nonmitogenic responses described above . In such cases, Erk protein kinases may act directly or indirectly on cyclins to arrest division and permit differentiation . The pathways influenced by ERK-like gene products in animal and yeast cells suggest that, depending on the downstream targets of substrates, transcriptional changes in a particular cell may occur to drive the cell cycle or, alternatively, withdrawal from the cell cycle may lead to specific differentiation events.(ABSTRACT TRUNCATED AT 250 WORDS)

Drugs, 1992 Feb, 43(2), 259 - 84
Terbinafine . A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in superficial mycoses; Balfour JA et al.; Terbinafine is an orally and topically active allylamine antifungal agent with a primarily fungicidal action in vitro . Its spectrum of in vitro activity includes a broad range of dermatophyte, filamentous, dimorphic and dematiaceous fungi, and some yeast species . In clinical trials, mycological and overall efficacy rates of around 90 and 80%, respectively, have been achieved in cutaneous dermatophyte infections (tinea corporis/cruris and tinea pedis) with terbinafine, administered either orally (250 or 500 mg/day) or topically (a 1% cream applied twice daily) . Similar rates of cure have been obtained with oral terbinafine in dermatophyte nail infections after relatively short treatment periods ranging from 3 to 12 months . Topical terbinafine has been effective in approximately 80% of patients with cutaneous candidiasis or pityriasis versicolor . Few comparative data have been published, but generally oral terbinafine appeared to be at least as effective as oral griseofulvin or ketoconazole in tinea corporis/cruris and more effective than griseofulvin in tinea pedis . Both oral and topical terbinafine have been very well tolerated in clinical trials to date, with only minor adverse effects reported . Although further research is required to establish the efficacy of terbinafine in comparison with other available therapies, as well as to fully clarify its tolerability profile, the early results obtained with terbinafine in superficial fungal infections are very encouraging . Terbinafine appears likely to become a first-line therapy for dermatophyte infections, particularly those affecting the nails.

Am J Physiol, 1992 Feb, 262(2 Pt 1), C396 - 404
Antibodies against the cystic fibrosis transmembrane regulator; Fuller CM et al.; Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480) . Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467) . These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with {gamma-32P}ATP in the presence of protein kinase A . Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system . Similar results were observed using a yeast CFTR expression system . In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein . These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.

Immunol Lett, 1992 Feb, 31(2), 189 - 97
Development of a Langerhans cell phenotype from peripheral blood monocytes; Rossi G et al.; Epidermal Langerhans cells (ELC) are definitively primed to differentiate into dendritic cells (DC) . It is unknown at what stage of monocyte development this priming occurs . In a culture system characterized by low paracrine stimulation, i.e . Iscove's modified Dulbecco medium (IMDM) with 2% FCS, we tested the ability of peripheral blood monocytes to turn to the route of the LC-DC lineage . In this system monocytes did not develop significant yeast cell phagocytosis, although mannose receptors were available . However, they became strong stimulators of mannan specific T cell proliferation . Phenotype development was analysed by flow cytometry using the monoclonal antibodies OKT6 (CD1a), IOT2 (HLA-DR), IOM2 (CD14) and the ligand Man-BSA-FITC . CD1a was the first marker which distinguished cultured monocytes from developing macrophages, obtained by addition of 8% human serum . Like cord blood Langerhans cells (CBLC) they internalized OKT6 in deep coated pits . They maintained a phenotype of monocyte derived Langerhans cells (MoLC) during eight days of in vitro culture, expressing CD1a, mannose receptors and HLA-DR and decreasing CD14, if left in their own conditioned medium . MoLC could be converted into macrophages by addition of human serum only within the first four days in vitro . Our data suggest that monocytes acquire an LC phenotype by autocrine stimulation.

Int J Radiat Biol, 1992 Feb, 61(2), 205 - 11
Induced accumulation of polyubiquitin gene transcripts in HeLa cells after UV-irradiation and TPA-treatment; Nenoi M; There are three species of ubiquitin gene transcripts in HeLa cells, termed UbA (approximately 0.7 kb), UbB (approximately 1.1 kb) and UbC (approximately 2.5 kb) . In the present report, the UbC transcript was shown to accumulate up to 2.5-fold after irradiation with UV light or treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) . The kinetic analysis indicated that the induced accumulation of UbC was rapid and transient; maximal accumulation of UbC was induced at 2.5 h after UV irradiation or 3 h after TPA treatment . Inhibition of a de novo protein synthesis by cycloheximide did not repress the induction of UbC after treatment with UV light and TPA . On the other hand, induction of UbA and UbB, in most cases, was not observed . UV-inducibility of human ubiquitin conjugating enzyme, E2(17k), was also tested . E2(17k) is a protein with high sequence similarity to the product of yeast DNA repair gene, RAD6 . While the RAD6 gene has been reported to be inducible by UV light, no change in E2(17k) gene transcript was observed after UV irradiation.

New Biol, 1992 Feb, 4(2), 91 - 6
Trans-regulation of homeotic genes in Drosophila; Kennison JA et al.; Homeotic genes of the Antennapedia and bithorax complexes control Drosophila development by encoding DNA-binding proteins that regulate the transcription of target genes . Because either the presence or absence of these DNA-binding proteins alters development, regulation of the spatial patterns of expression is crucial to normal development . Numerous gene products are required for properly regulated expression of Antennapedia and bithorax complex genes, but few (if any) are dedicated solely to the regulation of these genes . One of the pleiotropic activators of homeotic genes in Drosophila, the brahma gene, encodes a protein similar to a yeast protein that is required for transcriptional activation of multiple tightly regulated genes . Other components of this system may be conserved as well, suggesting that the biochemical basis for induced gene expression in single-celled organisms may have more in common with programmed developmental pathways in multicellular organisms than previously thought.

Transplantation, 1992 Feb, 53(2), 467 - 72
Immunohemopoietic effects of dietary nucleotide restriction in mice; Kulkarni AD et al.; The influence of dietary sources of nucleotides on host in vivo and in vitro immuno-hematologic responses in BALB/c (NCI) mice was studied . Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in popliteal lymph nodes undergoing proliferative response to syngeneic and allogeneic in vivo stimulation . Supplementation of a nucleotide-free (NF) diet with yeast RNA (NFR) or uracil (NFU) significantly enhanced the host PLN immune response as compared with NF and NF supplemented with adenine (NFA) diets . Levels of ADA and PNP enzymes in the PLNs increased with the alloimmune PLN response of host, and immunosuppression was associated with decreased ADA and PNP activities in lymphocytes following antigenic stimulation . The induction of these enzymes during immune response appears to require dietary sources of certain nucleotides . When bone marrow cells from control chow fed animals were cultured with supernatants (sups) from mitogen activated splenocytes of animals on each dietary group, NF sups significantly decreased (P less than 0.05) the BM proliferative response compared with the response observed with NFR sups, and similar to NFA or NFU sups . When stimulated with purified IL-3, NFR BM cells had higher levels of Thy1.2 or Lyt 1 surface markers as compared with other test groups . In the in vivo splenic colony formation-CFUs assay, spleens from NFR- and NFU-fed animals had a significantly higher number of colonies than spleens from NF- or NFA-fed mice . Thus, NF diet decreases both in vivo lymphoproliferation response to alloantigen and hemopoietic growth factor production, rendering the host splenic environment deficient for stem cell growth . These adverse effects are reversed by RNA supplementation of NF diet . These nutritional studies demonstrate a critical and regulatory role for dietary nucleotides in immunohemopoiesis.

Eur J Biochem, 1992 Feb 1, 203(3), 367 - 71
Identification of carrot cDNA clones encoding a second putative proliferating cell-nuclear antigen, DNA polymerase delta auxiliary protein; Hata S et al.; The proliferating cell-nuclear antigen (PCNA) plays a key role in the control of eukaryotic DNA replication . We have isolated two cross-hybridizing groups of cDNA encoding carrot homologs of PCNA . Sequence analysis and Southern-blot experiments showed that the cDNA were derived from two distinct genes . One corresponded to the typical PCNA, which is known to be highly conserved in eukaryotes from yeast to man; its mRNA is 1.2 kb in size and the calculated molecular mass of the protein is 29 kDa . The other encoded a larger PCNA homolog which has not previously been reported; the mRNA is 1.5 kb in size, the N-terminal three quarters (calculated molecular mass, 29 kDa) of the protein product is 88% identical at the amino acid level to the typical PCNA, but the protein has an extra C-terminal domain of 11 kDa . Both PCNA homologs were apparently coexpressed concomitant with somatic embryogenesis . The mRNA level of the novel homolog is 10-20% that of the typical PCNA in the embryos . The presence of the second putative PCNA may provide new insight into studies on the mechanism of DNA replication in eukaryotes.

Immunology, 1992 Feb, 75(2), 275 - 80
Protein kinase C activation modulates tumour necrosis factor-alpha priming of human neutrophils for zymosan-induced leukotriene B4 release; Petersen MM et al.; Neutrophil (PMN) activation by the yeast component zymosan involves the complement receptor type 3 (CD11b/CD18) . Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) augmented the zymosan-stimulated leukotriene B4 (LTB4) release from PMN, reaching a fourfold increase at 10(-9) M . Co-incubation of PMN with 10(-9) M rhTNF-alpha and staurosporine resulted in a further dose-dependent increase, which became significantly greater than a purely additive effect at a staurosporine concentration of 10 nM . This synergy was maintained at all doses of staurosporine tested . In addition, doses of phorbol 12-myristate 13-acetate (PMA) that do not activate protein kinase C (PKC) (below 10(-9) M) also augmented the zymosan-stimulated release of LTB4 . However, doses of PMA above 10(-9) M progressively inhibited the response to levels below that of zymosan alone . Staurosporine at 50 nM completely prevented, and 10(-9) M rhTNF-alpha partially but significantly (P less than 0.02 at 10(-8) M PMA, P less than 0.01 at 10(-7) M PMA) reversed, this high-dose PMA inhibition . PKC activation thus opposes the priming effect of rhTNF-alpha on neutrophils, while PKC inhibition may enhance the ability of rhTNF-alpha to prime PMN for zymosan activation . The combined effect of rhTNF-alpha and staurosporine suggests an intracellular synergy rather than simply a direct action due to increased zymosan receptor expression . Thus there appear to be mechanisms whereby the responses of neutrophils may be augmented without activating PKC . Indeed, kinase activation may even exert a degree of feedback control that is antagonized by rhTNF-alpha treatment.

J Am Diet Assoc, 1992 Feb, 92(2), 168 - 74
Food sources of energy, macronutrients, cholesterol, and fiber in diets of women; Krebs-Smith SM et al.; Data from the 1985 Continuing Survey of Food Intakes by individuals were used to calculate the contributions of individual foods to women's intakes of energy, protein, carbohydrate, fat, saturated fatty acids, cholesterol, and fiber . We separated nearly all food mixtures into their constituent ingredients, grouped the ingredients together with similar foods, and examined the contributions of those foods . Yeast breads that were neither whole grain nor higher fiber contributed about 7% of the energy to the diets, which made them the leading source of energy of the foods we examined . The leading sources of protein were animal products: poultry contributed approximately 12%, beef contributed about 19%, cheese contributed about 8%, and pork contributed about 6% . The various fats and oils were the greatest contributors to fat, and cheese was the chief source of saturated fatty acids . Eggs were the major source of cholesterol; they provided around 36% of the total . Two of the top three sources of carbohydrate--regular soft drinks and sugar--are composed entirely of simple sugars . Potatoes provided around 11% of the fiber, which made them the leading source of fiber . This article shows that the relative ranking of foods and the contribution of each food depend on the way food codes are combined . Therefore, citing one food as the major source of a particular food component without including documentation of how foods are combined can be misleading.

Eur J Biochem, 1992 Feb 1, 203(3), 353 - 60
Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2 . Evidence for two cooperative binding domains in p9CKShs2; Azzi L et al.; A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2----M transition of the cell cycle in all organisms . The yeast p13suc1 and p18CKS1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex . We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2 . Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a p34cdc2-binding site and that these two binding domains cooperate in establishing a stable p9CKShs2-p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1-Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively . No binding occurs in the absence of free peptide . This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKShs2 . The cyclin B subunit is not required for the formation of the p9CKShs2-p34cdc2 complex through these two binding domains . The implications of the existence of two cooperative p34cdc2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.

J Virol, 1992 Feb, 66(2), 922 - 9
Characterization of the Epstein-Barr virus BZLF1 protein transactivation domain; Flemington EK et al.; Initiation of the Epstein-Barr virus (EBV) lytic cycle is dependent on expression of the viral transactivator Zta, which is encoded by the BZLF1 gene . Described here is an initial mapping of the regions of Zta involved in activating transcription . The data indicate that the amino-terminal 153 amino acids of Zta are important for activity, and in particular the region from residues 28 to 78 appears to be critical for Zta function . However, other features of Zta may be important for activity since a Gal4-Zta chimeric protein, generated by fusing the amino-terminal 167 residues of Zta to the DNA binding domain of the yeast transactivator Gal4, transactivated a minimal promoter containing one upstream Gal4 binding site but was unable to exhibit synergistic transactivation when assayed with a reporter containing five upstream Gal4 binding sites.

J Biol Chem, 1992 Jan 25, 267(3), 1995 - 2005
TATA box-mediated polymerase III transcription in vitro; Mitchell MT et al.; We have defined conditions whereby a functional TATA box can mediate efficient in vitro transcription by RNA polymerase III . A TATA box is absolutely required for this reaction as a single-point mutation in this sequence completely abolishes transcription . Two protein components are also required: a HeLa cell phosphocellulose fraction (fraction B) and at least one other factor that can be supplied by various crude nuclear extracts or by HeLa cell phosphocellulose fractions C and D . The order of addition is critical; fraction B must be preincubated with the template DNA for TATA box-dependent polymerase III transcription to occur . Various TATA sequences are quite similar in their ability to mediate transcription by polymerases II and III . Despite the similarity in sequence requirements, fraction B does not appear to contain any detectable transcription factor (TF) IID activity, and TATA box-mediated polymerase III transcription does not appear to require TFIID in the form contained in phosphocellulose fraction D . It was recently reported that TFIID is required TFIID in the form contained in phosphocellulose fraction D . It was recently reported that TFIID is required for polymerase III transcription of the yeast and human U6 genes (Margottin, F., Dujardin, G., Gerard, M., Egly, J.-M., Huet, J., and Sentenac, A . (1991) Science 251, 424-426; Simmen, K . A., Bernues, J., Parry, H . D., Stunnenberg, H . G., Berkenstam, A., Cavallini, B., Egly, J.-M., and Mattaj, I . W . (1991) EMBO J . 10, 1853-1862) . We propose that fraction B may contain TFIID in a modified form that is not functional for polymerase II transcription.

Cell, 1992 Jan 24, 68(2), 323 - 32
Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B; Jessus C et al.; Activation of maturation-promoting factor at the onset of mitosis requires the tyrosine dephosphorylation of one of its components, the cdc2 protein kinase . cdc25 is the specific tyrosine phosphatase that activates cdc2 . We find that Xenopus oocytes contain a relative of cdc25, p72 . In Xenopus embryos the abundance of p72 does not oscillate during the cell cycle . However, p72 directly associates with cdc2-cyclin B in a cell cycle-dependent manner, reaching a peak at M phase . The M phase kinase that associates with p72 is catalytically active . These results suggest that the mechanism by which cdc25 triggers cdc2 activation involves a periodic physical association between cdc25 and the cyclin B-cdc2 complex and also that mitotic control can be affected by mechanisms other than transcriptional regulation of the cdc25 gene.

J Mol Biol, 1992 Jan 20, 223(2), 557 - 71
Ubiquitous soluble Mg(2+)-ATPase complex . A structural study; Peters JM et al.; We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases . Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits . For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits . Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx . 12 nm diameter; approx . 9 nm height) . In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx . 12 nm diameter; approx . 18.5 nm height) . Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique . Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation . A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented . From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 787 - 91
Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family; Palme K et al.; We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family . Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40% . Most amino acid residues showing identity are clustered in the GTP/GDP binding domain . In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well . Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs . The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells . The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers . The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.

J Immunol, 1992 Jan 15, 148(2), 562 - 7
A protective monoclonal antibody specifically recognizes and alters the catalytic activity of schistosome triose-phosphate isomerase; Harn DA et al.; mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner . Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice . Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate . After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced . Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI) . Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction . Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI . To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme.

Biochemistry, 1992 Jan 14, 31(1), 250 - 6
The molecular basis of cooperativity in protein folding . Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase; Freire E et al.; In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature {Griko, Y . V., Venyaminov, S . Y., & Privalov, P . L . (1989) FEBS Lett . 244, 276-278} . The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry . The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition . On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function . Analysis of the data indicates that at low temperatures the two domains behave independently of each other . The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains . These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds . This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK . It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.

Mycoses, 1992 Jan-Feb, 35(1-2), 17 - 21
Melanized and non-melanized multicellular form mutants of Wangiella dermatitidis in mice: mortality and histopathology studies; Dixon DM et al.; One melanized (Mc3) and one non-melanized (Mc3W) multicellular form mutant of W . dermatitidis were compared with parental wild type in NYLAR mice . Each mutant grows as multicellular (muriform-like) forms in vitro at 37 degrees C and as yeasts at less than or equal to 30 degrees C . Yeast cells of all three strains were injected intravenously at concentrations of 1 x 10(4), 1 x 10(6), 1 x 10(7), 3 x 10(7) and 1 x 10(8) cells/mouse in groups of 10 mice . There was no virulence difference between wild type and Mc3, with 100% mortality obtained with each strain at greater than or equal to 1 x 10(7) cells/mouse . In contrast, Mc3W was less virulent, with mortality being obtained only at 1 x 10(8) cells/mouse . Histopathological study of brains, lungs, livers and spleens of moribund mice revealed that both Mc3 and Mc3W persisted in tissue as muriform cells, and in some cases as yeast, pseudohyphal and hyphal forms . There was no major difference between Mc3 and Mc3W in terms of histopathological response . These data support the association between melanin and virulence in W . dermatitidis and provide a model for the study of muriform cells in vivo.

Henry Ford Hosp Med J, 1992, 40(3-4), 210 - 4
Isolation of YAC clones from the pericentromeric region of chromosome 10 and development of new genetic markers linked to the multiple endocrine neoplasia type 2A gene; Lairmore TC et al.; Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene . Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB . Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands . Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from end-clones . Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones . In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 alleles, PIC = 0.88), provide useful reagents for a polymerase chain reaction-based predictive genetic test that can be performed rapidly from small amounts of DNA.

J Biol Chem, 1992 Jan 5, 267(1), 210 - 7
Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR; Willem R et al.; The reversible conversion between D-glucose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphoglucoisomerase was studied by phase sensitive two-dimensional 13C-{1H} EXSY NMR spectroscopy at 150.869 and 125.759 MHz, using 13C-enriched substrates in the C2 position of the D-hexose 6-phosphates . The shape of the build-up curves of the cross-peaks associated with the 13C2 resonances of the alpha- and beta-anomers of both D-{2-13C}glucose 6-phosphate and D-{2-13C}fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between alpha-D-{2-13C}glucose 6-phosphate and beta-D-{2-13C}fructose 6-phosphate . Quantitative analysis of the build-up curves by three different methods allowed us to conclude that phosphoglucoisomerase not only selectively channels the latter isomerization but also considerably accelerates the anomerization of both D-hexose 6-phosphates . The rate constants of anomerization were indeed much higher in the presence than in the absence of enzyme . The major finding in the present study consists in the anomeric specificity of phosphoglucoisomerase toward the beta-anomer of D-fructose 6-phosphate both as a substrate and a product, contrary to previous proposals . This finding supports recent evidence suggesting the direct channelling of beta-D-fructose 6-phosphate from phosphoglucoisomerase to phosphofructokinase.

Genomics, 1992 Jan, 12(1), 52 - 7
The iduronate sulfatase gene: isolation of a 1.2-Mb YAC contig spanning the entire gene and identification of heterogeneous deletions in patients with Hunter syndrome; Palmieri G et al.; A recently isolated cDNA clone from the iduronate sulfatase (IDS) gene has been used both to seed a contig of overlapping yeast artificial chromosomes (YACs) and to investigate the molecular defect in patients with Hunter syndrome (MPS II) . Six YAC clones were found to span the IDS gene, and those and 14 other YACs were assembled into a 1.2-Mb contig around the gene in Xq27-q28 . The physical map of the region identifies several putative CpG islands, suggesting the presence of other genes in the vicinity . DNA from a patient with a translocation breakpoint in the gene also permitted the orientation of the contig in the chromosome . Southern analysis of DNA from 25 unrelated Italian Hunter syndrome patients revealed 4 with deletions or rearrangements in the IDS gene.

J Cell Biol, 1992 Jan, 116(2), 257 - 69
Characterization of the major hnRNP proteins from Drosophila melanogaster; Matunis EL et al.; To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster . cDNA clones of several D . melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes . These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D . melanogaster (Matunis et al., 1992, accompanying paper) . All of the proteins described here contain two amino-terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain . We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly . The sequences of the D . melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD-containing proteins has been conserved independently and has a different function . Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins . A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man . These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes . This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins.

Arch Virol, 1992, 122(1-2), 45 - 60
Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells; Naim HY et al.; The structure of the N-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo-beta-N-acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography . The results demonstrated that the glycoproteins decorating the virus shed from infected cells have N-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells . However, the E1 glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminal N-acetylglucosamine . The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at different N-glycosylation sites as in the case of E2, or at the same N-glycosylation site as in the case of E1 . In the presence of 1-deoxymannojirimycin, no structural heterogeneities in the glycan chains were found . This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 197 - 201
Differential expression of two distinct forms of mRNA encoding members of a dipeptidyl aminopeptidase family; Wada K et al.; We have identified two cDNAs encoding dipeptidyl aminopeptidase-like proteins (DPPXs) in both bovine and rat brains that have different N-terminal cytoplasmic domains but share an identical transmembrane domain and a long C-terminal extracellular domain . In both species, one of the cDNAs encodes a protein (designated DPPX-S) of 803 amino acid residues with a short cytoplasmic domain of 32 amino acids, and the other cDNA encodes a protein (designated DPPX-L) with a longer cytoplasmic domain--the bovine cDNA encodes 92 amino acids and the rat cDNA encodes 88 amino acids . The membrane topology of DPPX-S and -L is similar to that of other transmembrane peptidases, and DPPX-S share approximately 30% identity and 50% similarity with reported yeast and rat liver dipeptidyl aminopeptidase amino acid sequences, suggesting that DPPX is a member of the dipeptidyl aminopeptidase family . DPPX-S mRNA is expressed in brain and some peripheral tissues including kidney, ovary, and testis; in contrast, DPPX-L mRNA is expressed almost exclusively in brain . No transcripts for either form are found in heart, liver, or spleen . In situ hybridization studies show that the two transcripts have different distributions in the brain . DPPX-L mRNA is expressed in limited regions of brain with the highest level of expression in the medial habenula . More widespread expression is seen for DPPX-S mRNA . The differential distribution of mRNAs for the DPPX-S and -L suggests that these proteins are involved in the metabolism of certain localized peptides and that the cytoplasmic domain may play a key role in determining the physiological specificity of DPPX.

J Bacteriol, 1992 Jan, 174(2), 447 - 55
Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus; DiSanto ME et al.; A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus . The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized . The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4 . The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa . Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa . SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits . By using {1,3-3H}diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase . With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively . CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI) . The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate . The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme . Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus . The partial amino acid sequence of the carboxypeptidase was determined.

Crit Rev Biochem Mol Biol, 1992, 27(1-2), 129 - 55
Eukaryotic DNA replication; So AG et al.; The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level . Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses . New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced . In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins . In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.

J Virol, 1992 Jan, 66(1), 244 - 50
Regulation of human immunodeficiency virus enhancer function by PRDII-BF1 and c-rel gene products; Muchardt C et al.; The human immunodeficiency virus (HIV) enhancer element is important in the regulation of HIV gene expression . A number of cellular proteins have been demonstrated to bind to the NF-kappa B motifs in this element . The genes encoding several of these proteins, including members of the rel family and PRDII-BF1, have been cloned . We characterized the binding of proteins encoded by the human c-rel and PRDII-BF1 genes to HIV NF-kappa B motifs and related enhancer elements . Both the human c-rel protein and two proteins derived from the PRDII-BF1 gene by alternative splicing bound specifically to the HIV NF-kappa B motif and related enhancer elements found in the immunoglobulin kappa, class I major histocompatibility complex, and interleukin-2 receptor genes . To determine the role of these factors in regulating HIV gene expression, we fused the cDNAs encoding either of the two proteins derived by alternative splicing of the PRDII-BF1 gene or the c-rel gene to the DNA binding region of the yeast transcription factor GAL4 . GAL4 binding sites were inserted in place of the native HIV enhancer sequences in an HIV long terminal repeat chloramphenicol acetyltransferase construct . Cotransfection of these constructs revealed that c-rel was a strong activator of basal HIV gene expression but did not result in synergistic effects in the presence of tat . PRDII-BF1-derived cDNAs did not result in stimulation of either basal or tat-induced activated gene expression . These results indicate that multiple enhancer binding proteins may potentially regulate HIV in both a positive and negative manner.

Zentralbl Mikrobiol, 1992, 147(3-4), 283 - 7
Physiological study on riboflavin production by hydrocarbon-utilizing Candida guilliermondii Wickerham; Ghanem KM et al.; The production of riboflavin (vitamin B2) by Candida guilliermondii Wickerham cultivated on solar-containing medium was stimulated in the presence of corn oil (0.1 g%), arginine, phenylalanine (1 m mole/l) or CoSO4.7H2O (1000 micrograms/l) . On the other hand, emulsifying agents strongly inhibited yeast growth and vitamin production . Similarly, FeSO4.7H2O and MnSO4.4H2O at 50 and 200 micrograms/l levels, respectively, also showed inhibitory effect . Among the tested purines, xanthine enhanced vitamin production.

Life Sci, 1992, 50(25), 1963 - 72
Alanine and hyperosmolarity are responsible for the stimulation of cardiomyocyte glucose transport by samples containing a glucose tolerance factor; Fischer Y et al.; Low-molecular-weight, cationic samples, that were previously reported to contain a glucose tolerance factor, were obtained by partial purification from yeast extract . These samples increased the rate of glucose transport in isolated cardiomyocytes 2.0- to 2.5-fold . A further purification by gel filtration led to the separation of two active components that were identified as (i) L-alanine and (ii) an elevated osmolarity . Moreover, the effect of partially purified fractions (before gel filtration) (i) was decreased upon alanine depletion with alanine dehydrogenase and (ii) was mimicked by the additive action of alanine and of a hyperosmolar medium . These findings indicate that the effect of this partially purified material is not accounted for by a putative glucose tolerance factor . Interestingly, alanine elicited its effect at concentrations that correspond to physiological plasma values, which suggests that this amino acid might be involved in the regulation of glucose transport in cardiomyocytes . Furthermore, the effect of alanine was prevented by DL-cycloserine (1 mM) or aminooxyacetate (1 mM), but not by cycloheximide (35 microM), indicating that (a) transamination reaction(s), but not protein synthesis, is required.

Mol Carcinog, 1992, 5(3), 219 - 31
Elevated expression of the ribosomal protein S2 gene in human tumors; Chiao PJ et al.; Differential screening of a cDNA library was used to isolate genes differentially expressed in a nontumorigenic clone and a ras-transformed variant of the human teratocarcinoma cell line PA-1 . The RNA transcript for one of the cDNA clones that we identified was expressed at a 25-fold higher level in the ras-transformed PA-1 cells than in the nontumorigenic PA-1 cells . DNA sequence analysis of this clone showed that it had 86% nucleic acid homology to the mouse LLRep3 gene and only differed at a single amino acid codon (codon 198), which is changed from serine in LLRep3 to threonine in this cDNA clone . The rat ribosomal S2 protein is closely related to the yeast omnipotent informational suppressor SUP44, which encodes the yeast ribosomal protein S4; to the mouse protein LLRep3; and to the human cDNA clone we describe in this report . We therefore concluded that this clone codes for the human ribosomal S2 protein . In situ hybridization experiments revealed that expression of this gene was elevated in cultured human head and neck squamous cell carcinomas compared with normal keratinocytes . In situ hybridization experiments also demonstrated that expression of this gene was elevated in histological sections of human premalignant leukoplakia, head and neck squamous cell carcinomas, and colon and breast cancers compared with the adjacent normal tissues . S2 expression may be a useful diagnostic or prognostic marker for grading human tumors.

Biol Neonate, 1992, 61(2), 82 - 91
Characterization and developmental expression of binding sites for the transplacental iron transport protein, uteroferrin, in fetal hematopoietic tissues; Michel FJ et al.; In the pig, iron transport to the developing fetus during pregnancy involves, in part, uteroferrin (UF), a secreted progesterone-induced protein of the uterus . Neonatal pigs suffer from anemia, and the decrease in the synthesis of UF protein in late pregnancy was suggested to be partly responsible for this condition . To examine whether diminished capacity for UF uptake by pig fetuses may also contribute to neonatal anemia, binding sites for 125I-UF were examined in plasma membrane-enriched fractions of fetal liver and spleen, which are sites of fetal hematopoiesis . In addition, changes in the number of these binding sites as a function of fetal development were evaluated . Binding of 125I-UF to liver membrane fractions was displaced by intact UF greater than deglycosylated (aglyco) UF greater than ovalbumin, but not by yeast mannan . Scatchard analysis of radioligand binding showed the presence of a single class of binding sites with a dissociation constant of 10(-7) M . During fetal development and at postpartum (day 5), liver binding sites for UF remained invariant and displayed the same affinity . In contrast, the number of binding sites for UF in fetal spleen increased from midpregnancy to parturition and remained elevated in day 5 neonatal spleen . Affinity cross-linking of 125I-UF to liver membrane-associated binding sites and subsequent analysis by gel electrophoresis and autoradiography demonstrated a single labeled protein complex of Mr 58,000 and 87,000 under denaturing and nondenaturing conditions, respectively . The appearance of these bands was inhibited by intact UF, but not ovalbumin . The characteristics of the membrane-associated binding sites for UF differed from those of the mannose-related receptor previously described in reticuloendothelial cells of fetal liver . The invariant presence of UF binding components in sites of hematopoiesis during fetal development suggests that mechanism(s) unrelated to specific uptake of UF are responsible for neonatal anemia.

J Membr Biol, 1992 Jan, 125(2), 99 - 106
Targeting of proteins into the peroxisomal matrix; Subramani S; During the last few years much has been learned regarding signals that target proteins into peroxisomes . The emphasis in the near future will undoubtedly shift towards the elucidation of the mechanism of import . The use of mammalian and yeast cells deficient in peroxisome assembly and/or import (Zoeller & Raetz, 1986; Erdmann et al., 1989; Cregg et al., 1990; Morand et al., 1990; Tsukamoto, Yokota & Fujiki, 1990) should provide a handle on the genes (Erdmann et al., 1991; Tsukamoto et al., 1991) involved in these processes . This will have to be coupled with further development of in vitro systems which will permit the dissection of the steps in the translocation of proteins into peroxisomes . Though some progress has been made in the development of such assays (Imanaka et al., 1987; Small et al., 1987, 1988; Miyazawa et al., 1989), the fragility of peroxisomes and the absence of biochemical hallmarks of import (such as protein modifications or proteolytic processing) have hindered progress . Since peroxisomes exist in the form of a reticulum in mammalian cells (Gorgas, 1984), all peroxisome purification schemes (from mammalian cells at least) must undoubtedly rupture the peroxisomes, which then reseal to form vesicular structures . Additionally, the reliance on the latency of catalase alone as a major criterion for the integrity of peroxisomes ignores the fact that many other matrix proteins leak out of peroxisomes at vastly different rates during purification of the organelles (Thompson & Krisans, 1990) . In view of these problems, the development of peroxisomal transport assays with semi-intact cells would also constitute an important advance.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenetics, 1992, 35(3), 183 - 9
A continuous restriction map from HLA-E to HLA-F . Structural comparison between different HLA-A haplotypes; el Kahloun A et al.; The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H) . By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A . The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region . We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31) . The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.

Am J Ind Med, 1992, 21(2), 177 - 91
Immunological and respiratory changes in animal food processing workers; Zuskin E et al.; A group of 35 men employed in the processing of animal food was studied to assess the relation between respiratory findings and immunological status . The most frequent positive skin prick reactions to occupational allergens were to fish flour (82.9%), followed by carotene (77.1%), corn (65.7%), four-leaf clover (62.9%), sunflower (54.3%), chicken meat (31.4%), soy (28.6%), and yeast (22.7%) . Increased total IgE serum levels were found in 14/35 (40.0%) animal food workers compared to 1/39 (2.6%) in a healthy population (p less than 0.01) . A significantly higher prevalence of chronic respiratory symptoms was found among the exposed workers compared to control workers . There was however, no significant difference in the prevalence of chronic respiratory symptoms between animal food workers with positive and negative skin tests to house dust or to fish flour or among those with increased or normal IgE (except for dyspnea) . The frequency of acute symptoms associated with the work shift was high among the animal food workers but similar by immunological status . There were significant mean across-shift reductions for all ventilatory capacity tests, being particularly pronounced for FEF25 . Workers with positive skin tests to fish flour antigen had significantly larger across-shift reductions in FEF25 than workers with negative skin reactions . An aqueous extract of animal food dust caused a dose-related contractile response of isolated guinea pig tracheal muscle in vitro . Our data suggest that, in addition to any immunological response animal food dust may produce in vivo, it probably also causes direct irritant or pharmacological reactions on the airways as suggested by our in vitro data.

J Basic Microbiol, 1992, 32(1), 21 - 7
Purification and characterization of an inducible L-lysine: 2-oxoglutarate 6-aminotransferase from Candida utilis; Hammer T et al.; L-Lysine:2-oxoglutarate 6-aminotransferase (EC 2.1.6.36) was purified 202-fold from the yeast Candida utilis . The subunit Mr estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 40 kDa . The Mr of the native enzyme was estimated to be 83 kDa by gel filtration, suggesting a dimeric structure . The enzyme exhibits absorption maxima at 280, 340 and 420 nm, and binds 2 mol pyridoxal-5-phosphate/mol of the native enzyme . The aminotransferase has a maximum activity at pH 8.5 and at 4 degrees C . 2-Oxoglutarate is the best amino acceptor with L-lysine as amino donor . Lower activity is observed with oxaloacetate (38%), pyruvate (19%) and 2-oxoadipate (7%) as acceptor or with L-thialysine (13%) as donor . The Km values are 2.5 mM for L-lysine, 3.8 mM for 2-oxoglutarate and 0.015 mM for pyridoxal-5-phosphate . The enzyme activity is induced in cells grown in the presence of L-lysine.

Zentralbl Mikrobiol, 1992, 147(1-2), 86 - 91
Production of ochratoxins by some Egyptian Aspergillus strains; el-Shayeb NM et al.; In all the investigated Aspergillus ochraceus and A . alliaceus strains the maximum quantities of ochratoxins produced on yeast extract-sucrose (YES) medium exceeded that detected on wheat solid medium except A . ochraceus 67 . The shake cultures lowered ochratoxins formation . The maximum yields of ochratoxins did not coincide with maximum fungal growth . In most A . strains investigated, the total ochratoxin contents of the culture filtrates highly exceeded that of the mycelia.

Acta Obstet Gynecol Scand Suppl, 1992, 155, 39 - 54
Intracellular actions of steroid hormones and their therapeutic value, including the potential of radiohalosteroids against ovarian cancer; Holt JA et al.; The biological activities of steroid hormones are effected via intracellular receptors . The receptors are part of a ligand-activated family of transcription regulator proteins that are critical for steroid-regulated cell differentiation . With recombinant cDNA technology, yeast and cultured animal cells can be made to express mammalian cDNA steroid receptors from cDNA clones that contain deletions and substitutions . Among the leading problems addressed in these models is the characterization of sequences that promote association or interaction with other transcription regulating molecules, including oncogene products . Recently it has been found that heat shock proteins may serve not only to stabilize the receptor proteins but also to precondition the activation imparted by ligand binding . Aberrant receptor proteins can be found in ovarian cancer . Whether aberrant receptor proteins are associated with transformation in general or with a variable clinical response to steroidal or anti-steroidal therapy is not known . Even after chemotherapy, steroid receptors are expressed in the metastases of ovarian cancers seen clinically, and they may have potential uses for localization and treatment of receptor-rich cancers . Radioligand pharmaceuticals appropriate for imaging or for site-directed radiocytotoxicity can be sequestered to the nuclei of receptor-rich cancers . Initial clinical imaging and therapy trials with such pharmaceuticals have been approved and begun . In the use of halogenated estrogen radiopharmaceuticals, liver metabolism and enterohepatic recirculation are important considerations . Ascites prolongs retention of a radiohalogenated estrogen in the abdominal cavity . Distant metastases have been localized with {123I}-estrogen in breast cancer patients in pre-operative procedures . Receptor-mediated cytotoxicity occurs when estrogen receptor radioligand pharmaceuticals that are Auger electron emitters are used in vitro.

Mol Carcinog, 1992, 5(4), 270 - 7
Evidence for coincident mutations in human lymphoblast clones selected for functional loss of a thymidine kinase gene; Li CY et al.; A mitotic "recombination-competent state" inducible by x-irradiation is thought to exist in yeast . We sought evidence for such a process in mammalian cells by examining the occurrence of mutations at unlinked loci in clones derived from a human lymphoblast cell line . A total of 169 independent clones that arose spontaneously or after exposure to x-rays or ethyl methanesulfonate were selected for new somatic mutations at the thymidine kinase gene on chromosome 17q . They were subsequently screened for coincident mutations by use of variable-number-of-tandem-repeat probes located on different chromosomes . Three coincident mutations were positively identified by Southern analysis on chromosomes 7 and 14; they included one that produced a new allele and two that caused loss of allele heterozygosity . Densitometric analysis of the latter two indicated the presence of two copies of the remaining allele . Several possible coincident genetic events were also observed on chromosome 17 . These findings revealed a coincident mutant fraction of about 10(-2)/cell, whereas the expected mutation fraction at these loci is less than 10(-4)/cell . These results may thus provide the first molecular evidence that a "global" mutational process capable of inducing genetic instability exists in mammalian cells.

Methods Enzymol, 1992, 219, 261 - 7
Use of two-stage incubations to define sequential intermediates in endoplasmic reticulum to Golgi transport; Davidson HW et al.; Identification of the temporal requirement for components through the use of two-stage incubations is valuable in dissecting the overall transport reaction into steps relevant to vesicle fission and those related to vesicle fusion . In the context of semiintact mammalian cells in which a functional vesicle intermediate has not been detected, components playing a role in targeting are presently difficult to identify . However, the two-stage incubations are particularly powerful when either the donor or acceptor compartments can be manipulated independently, as is the case for intra-Golgi transport using enriched Golgi fractions or in the case of ER-to-Golgi transport in perforated yeast, in which a vesicle intermediate can be physically isolated.

Subcell Biochem, 1992, 18, 131 - 87
Molecular karyotype analysis in Leishmania; Bastien P et al.; The advent of pulsed field electrophoresis has allowed a direct approach to the karyotype of Leishmania . The molecular karyotype thus obtained is a stable characteristic of a given strain, although minor modifications may occur during in vitro maintenance . Between 20 and 28 chromosomal bands can be resolved depending on the strain, ranging in size from approximately 250 to 2600 kb . The technique has revealed a striking degree of polymorphism in the size and number of the chromosomal bands between different strains, and this seems independent of the category (species, zymodeme, population) to which the strains belong . It appears that only certain strains originating from the same geographic area may share extensive similarities . This polymorphism can largely be accounted for by chromosome size variations, which can involve up to 25% of the chromosome length . As a result, homologous chromosomes can exist in versions of markedly different size within the same strain . When this occurs with several different chromosomes, the interpretation of PFE patterns appears difficult without prior identification of the size-variable chromosomes and of the chromosome homologies . DNA deletions and amplifications have been shown to account for some of these size modifications, but other mechanisms are probably involved; nevertheless, interchromosomal exchange does not seem to play a major role in these polymorphisms . These chromosomal rearrangements, yet in an early stage of characterization, exhibit two relevant features: they seem (1) to affect essentially the subtelomeric regions and (2) to occur in a recurrent nonrandom manner . Chromosomal rearrangements sharing the same characteristics have been identified in yeast and other protozoa such as Trypanosoma and Plasmodium . The significance of this hypervariability for the biology of the parasite remains unknown, but it can be expected that such mechanisms have been maintained for some purpose; genes specifically located near chromosome ends might benefit from rapid sequence change, alternating activation, or polymorphism of expression . The chromosomal plasticity could represent a general mode of mutation in these parasites, in parallel with genetic exchange which may be uncommon in nature . The molecular characterization of these rearrangements, the identification of each chromosome with the help of physical restriction maps and linkage maps, and the collation of such data on a number of strains and species should allow a significant progress in the understanding of the genetics of Leishmania, in particular as regards ploidy, generation of phenotypic diversity, and genome evolution . Finally, like other models, this is susceptible to improve our knowledge of DNA-DNA interactions and of the chromosome functional structure and dynamics.

Nahrung, 1992, 36(5), 485 - 9
Mycotoxic flora and mycotoxins in smoke-dried fish from Sierra Leone; Jonsyn FE et al.; Examination of 20 samples of smoke-dried fish of the Ethmolosa sp . commonly called "Bonga", from homes and markets in Njala (Sierra Leone) revealed the presence of 4 Aspergilli species: A . flavus Links ex Fries, A . ochraceus Wilhelm, A . tamarii Kita and A . niger van Tieghem . Fresh or properly preserved smoke-dried fish showed no signs of fungal contamination . Mouldy fish extracts contained varying amounts of aflatoxins B1, G1, G2 and ochratoxin A . Isolates of A . flavus grown on yeast extract sucrose (YES) medium, produced considerable amounts of aflatoxin B1 and G1 and trace amounts of G2 . On YES medium A . ochraceus produced large amounts of ochratoxin A but no penicillic acid.

Int J Vitam Nutr Res, 1992, 62(3), 244 - 7
Effect of nitrite ingestion on the bioavailability of folate in the rat; Hoppner K et al.; The effect of nitrite ingestion on the intestinal deconjugation and absorption of folic acid (PGA) and brewers yeast folate was investigated using a rat bioassay and liver folate uptake as the response parameter . Male weanling Sprague Dawley rats were depleted on a low folate AIN-76A formulated basal diet for 21 days . During a 14 day repletion period, folic acid (PGA) and brewers yeast were added to provide 0.25, 0.5 and 1.0 mg of folate per kg of diet . Potassium nitrite was administered as part of the diet at 0.5% . All diets were made isonitrogenous and isocaloric . Based on a parallel line assay, the relative bioavailability of folate in the brewers yeast diet (109) was significantly higher than in the standard diet (PGA = 100) . When combined with nitrite, the relative bioavailability of the PGA diet was not significantly different (101), while that of the brewers yeast diet was significantly lower than the standard diet . Concomitant ingestion of nitrite significantly reduced the bioavailability of brewers yeast folate but not that of PGA in rats . This appeared to be a direct effect of oxidation by nitrite on the more susceptible substituted folates in brewers yeast.

Scand J Clin Lab Invest Suppl, 1992, 210, 5 - 22
The aspartic proteases; Szecsi PB; The Aspartic proteases (EC 3.4.23) are a group of proteolytic enzymes that share the same catalytic apparatus . Members of the aspartic protease family can be found in different organisms, ranging from humans to plants and retroviruses . The best known sources of aspartic proteases are the stomach of mammals, yeast and fungi, with porcine pepsin as the proto type . The aim of this review is to summarize some of the characteristics of the aspartic protease family.

Int Rev Cytol, 1992, 141, 217 - 32
Transcription and replication of animal mitochondrial DNAs; Clayton DA; The development of in vitro transcription and replication systems has allowed the identification of promoter sequences and origins of replication for several animal mtDNAs . As a consequence, the necessary reagents and basic information are available to permit the characterization of transacting factors that are required for transcription and replication . All of the animal trans-acting species purified at this time are known or reasoned to be nuclear gene products . There is now the opportunity to learn how these nuclear genes are regulated and the mechanisms that are utilized for the import of their products into the organelle . With regard to import, the human transcription factor mtTF1 appears to have an amino-terminal sequence characteristic of other imported mitochondrial proteins (Parisi and Clayton, 1991) . An interesting issue is the degree to which fundamental features of mtDNA replication and transcription are conserved between species . With regard to animal mtDNAs, there is very little in the way of conservation of DNA sequence at the promoters and origins of replication . The exceptions to this are the presence of a characteristic stem-loop L-strand origin of replication sequence in vertebrates {except for chicken mtDNA (Desjardins and Morais, 1990)} and the general presence of CSBs II and III (and to a lesser extent CSB I) in most higher animal mtDNAs . Because mtDNA promoters are not highly conserved, it is perhaps not surprising that general cross-species transcription does not occur, except for very limited examples of closely related species and sequences (Chang et al., 1985b) . Using crude mtRNA polymerase holoenzyme preparations, there is no specific transcriptional initiation when proteins from human mitochondria are used with mouse mtDNA promoter templates, and vice versa . However, in contrast to this overall observation, purified fractions of human or mouse mtTF1 can be exchanged and shown to function across species boundaries (Fisher et al., 1989) . The ability of mitochondrial mtTF1-type proteins to operate across even greater evolutionary distances was suggested by the ability of human and yeast proteins to recognize some mitochondrial promoter sequences in common (Fisher et al., 1992) . More recent studies suggest that human mtTF1 can substitute for its yeast homolog in vivo, and thereby perform at least the most critical functions required to maintain yeast mtDNA in the cell (M.A . Parisi, B . Xu, and D.A . Clayton, submitted for publication) . The other sites of conserved macromolecular interactions are related to the two origins of DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)

Arch Virol Suppl, 1992, 4, 154 - 5
Kinetics of anti-HBs after hepatitis B vaccination: a comparison of two recombinant and one plasma-derived vaccines; Gesemann M et al.; Geometric mean titers were determined for three groups of medical students who had been vaccinated against hepatitis B with two different yeast-derived recombinant vaccines and one plasma-derived vaccine . The antibody kinetics for the three groups were similar over a period of 4 years . A formula for prediction of titers from the post-booster anti-HBs concentration is provided.

Arch Virol Suppl, 1992, 4, 147 - 53
Immunogenicity and safety of a recombinant hepatitis B vaccine produced in mammalian cells and containing the S and the preS2; Corradi MP et al.; A group of 273 health care workers, at risk of HBV infection, underwent vaccination with recombinant HBsAg produced in mammalian cells and containing protein sequences coded by both the S and pre-S2 regions (Genhevac B) . Preliminary results show that a very early pre-S2 response occurred which may be useful in post-exposure prophylaxis . This observation, in addition to reduced influence by the vaccination protocol, provides grounds for optimism in spite of the fact that the efficiency spectrum of this vaccine was not superior to that of recombinant vaccines produced in yeast.

Acta Physiol Scand Suppl, 1992, 607, 131 - 6
The regulatory domain of fungal and plant plasma membrane H(+)-ATPase; Serrano R et al.; The activity of fungal and plant plasma membrane H(+)-ATPases seems to be regulated by modulation of the interaction of an inhibitory domain at the C-terminus with the active site . In the yeast ATPase, a mutation at the active site (Ala547- > Val) and a deletion of the C-terminus result in constitutive activation . A double Ser911- > Ala, Thr912- > Ala mutation at the C-terminus (defining putative phosphorylation sites) locks the enzyme in the inhibited state and can be suppressed by the Ala547- > Val mutation at the active site . This provides genetic evidence for domain interaction . In plant ATPase, proteolytic removal of the C-terminus also results in constitutive activation . A peptide covering a region of the plant C-terminus with homology to the yeast C-terminus inhibits the truncated plant ATPase . This suggests similar regulatory mechanisms in fungal and plant ATPases.

Cancer Surv, 1992, 14, 31 - 40
The importance of normal and abnormal oestrogen receptor in breast cancer; McGuire WL et al.; We have used the screening techniques of CMC and SSCP of selected PCR fragments, and also gel retardation assays, to discover a number of ER variants in clinical breast cancer tissues . We have found base pair insertions, transitions, and deletions, and deletions of exons 3, 5 and 7 . Using a yeast transactivation assay, we have discovered receptors with outlaw function consisting of both dominant positive receptors that were transcriptionally active in the absence of oestrogen and dominant negative receptors that were transcriptionally inactive themselves but prevented normal ER function . Future efforts should focus in particular on such dominant positive and dominant negative variants . With regard to positive variants, we should like to know whether they stimulate tumour growth and, if so, whether they can be turned off . With regard to dominant negative variants, we should like to determine whether they can inhibit tumour growth and, if so, whether they can be turned on.

J Clin Lab Anal, 1992, 6(5), 315 - 8
G test, a new direct method for diagnosis of Candida infection: comparison with assays for beta-glucan and mannan antigen in a rabbit model of systemic candidiasis; Miyazaki T et al.; An indirect method to measure beta-glucan, a major structural component of yeast cell walls, is available, but has the disadvantage of requiring the combined use of two assays . Recent reports describe the fungal index, which measures the difference between the conventional limulus test, in which factors C and G react with endotoxin and beta-glucan, and a new endotoxin-specific test, in which only factor C reacts with endotoxin . The G test was developed as a direct method to measure beta-glucan, and contains only factor G reacting with beta-glucan alone . In this study, the G test was examined in sera of rabbits with experimental systemic candidiasis, and compared with the fungal index and mannan assay . The G test showed positive in all rabbits with systemic candidiasis faster and with higher titers than with the fungal index . Three rabbits with fulminant systemic candidiasis showed higher levels of reactivity with the G test and the fungal index than two rabbits with mild reactions . Mannan was positive by at least one serum in four of five rabbits by the latex agglutination test, and there was a good correlation between these assays . The G test is a good serodiagnostic method for the detection of candidiasis.

Zhonghua Zhong Liu Za Zhi, 1992 Jan, 14(1), 42 - 4
{Influence of dietary selenium level on immune function of rats with esophageal tumors induced by methylbenzylnitrosamine (NMBzA)}; Zhu M; The influence of dietary selenium of the incidence of esophageal tumor induced by NMBzA and the immune function during carcinogenesis were studied in rats fed with Torula yeast diet and survived for 18 weeks . The incidences of esophageal tumors were statistically not significant among rats on normal, high and low selenium intake (P greater than 0.05) . The level of plaque forming cells (PFC), delayed type hypersensitivity (DTH), natural killer cell activity (NK) were significantly higher in the high selenium diet group than those of the low selenium diet group (P less than 0.05) . The authors believe that the modulation of dietary selenium can alter the immune function of animals during carcinogenesis but the anticarcinogenic effect of selenium still needs further study.

Mol Carcinog, 1992, 6(2), 83 - 7
Alternative splicing of neurofibromatosis type 1 gene transcript in malignant brain tumors: PCR analysis of frozen-section mRNA; Mochizuki H et al.; The neurofibromatosis type 1 (NF1) gene encodes a 360-residue region showing significant homology to the catalytic domains of both mammalian GTPase-activating protein (GAP) and yeast IRA protein . The product of the GAP-related domain of the NF1 gene (NF1-GRD) has been shown to stimulate ras GTPase and consequently to inactivate ras protein . We previously reported that the NF1-GRD has two types of transcripts, type I and type II, which are generated by an alternative splicing mechanism, and that the differential splicing of the NF1-GRD may be related to differentiation of neuroectodermal cells . Here we examined the differential expression of type I and type II transcripts of NF1-GRD in clinical samples of supratentorial malignant brain tumors by the RNA-polymerase chain reaction (PCR) method using frozen tissue sections . Our observations revealed that normal cerebrum predominantly expressed the type II NF1-GRD transcript, whereas primitive neuroectodermal tumors predominantly expressed the type I transcript . Additionally, although the type I/type II ratio in astrocytomas varied widely among tissue samples, all glioblastomas showed higher type I/type II ratios than adjacent brain samples . The RNA-PCR analysis using frozen tissue sections is a useful and sensitive method for detecting genetic markers in clinical tissue samples.

Nephron, 1992, 61(3), 284 - 6
Hepatitis B vaccination in dialysis centres: advantages and limits; Rapicetta M; A review is presented on the results achieved in HBV immunization of hemodialysis patients with aluminum-adsorbed, subunit plasma-derived and yeast-derived vaccines . The main host-related and immunization-related causes of low response are discussed . Various possibility to improve the vaccine performance through appropriate dosage and schedule, as well as immunopotentiating procedures are reported.

DNA Seq, 1992, 2(4), 265 - 7
Primary structure and in vitro expression of the N . crassa phosphoglycerate kinase; Azevedo JE et al.; The primary structure of 3-phosphoglycerate kinase (PGK) from Neurospora crassa was determined by sequencing a full-length cDNA . The deduced 418 amino acids protein shows a considerable identity to PGKs of other organisms with all the residues thought to be important for the function of the yeast enzyme conserved . The cloned PGK cDNA could be efficiently expressed in vitro resulting in a product with the expected molecular weight.

Mol Gen Genet, 1992 Jan, 231(2), 233 - 42
Extreme heterogeneity of Ty1-copia group retrotransposons in plants; Flavell AJ et al.; We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants . All eight species studied contain reverse transcriptase fragments from Ty1-copia group retrotransposons . Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75% . Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila . The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants . Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species . We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.

Trends Genet, 1992 Jan, 8(1), 27 - 32
Conservation and evolution of transcriptional mechanisms in eukaryotes; Guarente L et al.; Eukaryotic transcriptional activators play key roles in controlling cell growth and specifying embryonic development . These activators can stimulate promoters from distances up to tens of kilobases by a mechanism that is remarkably conserved in eukaryotes ranging from yeast to humans . Although the primary sequence of certain activators has also been conserved in widely divergent organisms, the regulatory roles that these factors play have been altered over evolution to fit the specific needs of the host.

Trends Biotechnol, 1992 Jan-Feb, 10(1-2), 19 - 22
Model genomes: the benefits of analysing homologous human and mouse sequences; Hood L et al.; The human genome initiative has provided the motivating force for launching sequencing projects suitable for testing various DNA-sequencing strategies, as well as motivating the development of mapping and sequencing technologies . In addition to projects targeting selected regions of the human genome, other projects are based on model organisms such as yeast, nematode and mouse . The sequencing of homologous regions of human and mouse genomes is a new approach to genome analysis, and is providing insights into gene evolution, function and regulation which could not be determined so easily from the analysis of just one species.

Henry Ford Hosp Med J, 1992, 40(3-4), 199 - 204
Localization of the gene for MEN 2A; Lichter JB et al.; The search for the gene that causes the multiple endocrine neoplasia type 2A (MEN 2A) syndrome is entering a new phase . Genetic linkage studies have localized the gene to the pericentromeric region of chromosome 10 . The statistical portion of mapping the gene for MEN 2A is nearly complete and now classical molecular biological/gene mapping techniques will be employed . We have used fluorescence in situ hybridization to estimate the size of the MEN2A region to be about 2 to 5 mb, using some liberal assumptions; at worst the region should contain no more than about 10 mb of non-alphoid DNA . Our mapping panels (meiotic recombinant and radiation reduced hybrid) give consistent orders of markers in this small region . We describe our initial attempts to clone the region using yeast artificial chromosomes.

Acta Derm Venereol, 1992, 72(1), 72 - 3
Anthralin is a potent inhibitor of pityrosporum orbiculare/ovale in vitro; Bunse T et al.; Two strains of Pityrosporum orbiculare/ovale were grown in a liquid medium and exposed to different concentrations of the imidazoles ketoconazole and clotrimazole as well as anthralin, liquor carbonis detergens and salicylic acid . With regard to growth inhibition of yeast cells, the efficacies of anthralin and the imidazoles were similar, a half-maximal inhibition being achieved with an anthralin concentration of 7 mg/l . Liquor carbonis detergens and salicylic acid also inhibited growth of Pityrosporum orbiculare/ovale, but only at much higher concentrations . The response to salicylic acid was mainly due to its acid pH.

Am J Hum Genet, 1992 Jan, 50(1), 56 - 64
Mapping of 262 DNA markers into 24 intervals on human chromosome 11; Tanigami A et al.; We have extended our mapping effort on human chromosome 11 to encompass a total of 262 DNA markers, which have been mapped into 24 intervals on chromosome 11; 123 of the markers reveal RFLPs . These clones are scattered throughout the chromosome, although some clustering occurs in R-positive bands (p15.1, p11.2, q13, and q23.3) . Fifty-two of the markers were found to contain DNA sequences conserved in Chinese hamster, and some of these 52 also cross-hybridized with DNA from other mammals and/or chicken . As the length of chromosome 11 is estimated at nearly 130 cM, the average distance between RFLP markers is roughly 1 cM . The large panel of DNA markers on our map should contribute to investigations of hereditary diseases on this chromosome, and it will also provide reagents for constructing either fine-scale linkage and physical maps or contig maps of cosmids or yeast artificial chromosomes.

Mem Inst Oswaldo Cruz, 1992, 87 Suppl 3, 85 - 9
A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence; Kemp DJ et al.; Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis . Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and the selected cells produce gametocytes . The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region . We have isolated several markers for the deleted region and are currently using a YAC-P . falciparum library to investigate this region of the genome in detail.

Ciba Found Symp, 1992, 170, 130 - 40; discussion 140-6
Protein phosphatases and cell division cycle control; Yanagida M et al.; Fission yeast has at least ten protein phosphatase genes that appear to play distinct roles in cell cycle control . Because of functional overlap, a clear lethal phenotype can be obtained only after multiple genetic alterations . Cells that have lost the protein phosphatase 1 (PP1)-like dis2/sds21 phosphatase activities prematurely enter mitosis and remain in a defective mitotic state with high H1 kinase activity and without sister chromatid disjunction . The same phenotype can be obtained in the presence of hydroxyurea . Overexpression of PP1-like phosphatase, on the other hand, delays the entry into mitosis . Cells that have lost PP2A-like ppa2 phosphatase activity also prematurely enter mitosis with a reduction in cell size . This semi-wee phenotype is enhanced in delta ppa2 mutants treated with the phosphatase inhibitor, okadaic acid . Genetic interactions between ppa2 and mitotic regulators suggest that ppa1/ppa2 phosphatase may directly or indirectly inhibit p34cdc2/cyclin kinase . Thus both PP1- and PP2A-like phosphatases in fission yeast may negatively regulate entry into mitosis . The major property of the dis2/sds21 mutant which is distinct from those of the ppa2/ppa1 mutant is its failure to inactivate the p34cdc2/cyclin complex after entry into mitosis . A novel phosphatase regulator encoded by sds22+ binds to dis2 phosphatase and controls the substrate specificity which appears to become essential in the progression from metaphase to anaphase.

Int J Cancer Suppl, 1992, 7, 28 - 32
CTLA-4 and CD28: similar proteins, neighbouring genes; Balzano C et al.; Subtractive cloning and screening yielded a cDNA clone corresponding to a molecule expressed in activated T cells, called CTLA-4 . At the protein level, CTLA-4, a single-V-domain member of the immunoglobulin superfamily, was found very homologous to the lymphocyte activation molecule CD28 . In particular, the hinge region included the hexamer MYPPPY, completely conserved for both molecules and in mice and humans . By immunizing mice with a human CTLA-4 peptide, an anti-CTLA-4 monoclonal antibody (MAb) was obtained, which enabled to establish the MW of the protein (26 and 40 kDa under reduced and non-reduced conditions respectively) and its preliminary tissue distribution . Also, CTLA-4 and CD28 were very similar at the message and at the gene structure level . The corresponding genes had previously been found to co-map on mouse chromosome IC and on human chromosome 2q33 . We show that they can be found on the same yeast artificial chromosomes bearing human genomic DNA, and that they are 25 to 150 kb apart . These marked homologies and gene proximity strongly suggest that CTLA-4 and CD28 are the direct products of a duplication event, and raise the question of the function of CTLA-4.

Appl Biochem Biotechnol, 1992 Jan-Mar, 32, 159 - 70
Immobilization of invertase through its carbohydrate moiety on Ocimum basilicum seed; Melo JS et al.; Yeast invertase, a glycoprotein, was covalently coupled to Ocimum basilicum seeds either through its protein or carbohydrate moiety . Of the various methods investigated, binding of the enzyme through its carbohydrate moiety resulted in the retention of considerably higher amounts of enzyme activity . Immobilized invertase showed a shift in the pH optimum toward the alkaline side without appreciable change in temperature optimum . However, the immobilized preparation was more thermostable than the free enzyme . Invertase bound to the seeds could be used repeatedly for the hydrolysis of sucrose syrups in a batch process without appreciable loss in activity . The seeds could serve as an inexpensive, ready-to-use, natural pellicular polysaccharide support for immobilizing enzymes.

Biol Cell, 1992, 75(2), 139 - 43
Higher eucaryotic cdc25 proteins are structurally related to phosphoseryl/threonyl protein phosphatases; Belle R et al.; cdc25 proteins are universally involved in the control of cell division . Using an original method of sequence analysis, cdc25 proteins from different sources were compared to protein phosphatases . Protein phosphatases could clearly be characterized as two distinct protein families, the phospho-seryl/threonyl phosphatases, and the phospho-tyrosyl phosphatases . None of the cdc25 proteins analyzed fitted with the phospho-tyrosyl phosphatases, indicating that if they indeed possess this biochemical activity, they form a distinct phsophatase protein group . Unexpectedly, higher eucaryotic cdc25 proteins (from human and fly) were found to be structurally related to phospho-seryl/threonyl phosphatases . These results fit well with expected function of the proteins, associated solely in higher eucaryotes, to dephosphorylation of threonine in the cell cycle control protein cdc2.

EMBO J, 1992 Jan, 11(1), 291 - 303
Retroviral integration into minichromosomes in vitro; Pryciak PM et al.; We describe here the use of chromatin as a target for retroviral integration in vitro . Extracts of cells newly infected with murine leukemia virus (MLV) provided the source of integration activity, and yeast TRP1ARS1 and SV40 minichromosomes served as simple models for chromatin . Both minichromosomes were used as targets for integration, with efficiencies comparable with that of naked DNA . In addition, under some reaction conditions the minichromosomes behaved as if they were used preferentially over naked DNAs in the same reaction . Mapping of integration sites by cloning and sequencing recombinants revealed that the integration machinery does not display a preference for nucleosome-free, nuclease-sensitive regions . The distributions of integration sites in TRP1ARS1 minichromosomes and a naked DNA counterpart were grossly similar, but in a detailed analysis the distribution in minichromosomes was found to be significantly more ordered: the sites displayed a periodic spacing of approximately 10 bp, many sites sustained multiple insertions and there was sequence bias at the target sites . These results are in accord with a model in which the integration machinery has preferential access to the exposed face of the nucleosomal DNA helix . The population of potential sites in chromatin therefore becomes more limited, in a manner dictated by the rotational orientation of the DNA sequence around the nucleosome core, and those sites are used more frequently than in naked DNA.

Genes Dev, 1992 Jan, 6(1), 105 - 16
Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator; Prakash SS et al.; The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT . The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410) . Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified . Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric . The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect . A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer . No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked . Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan . Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional . Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function . The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

Genetica, 1992, 87(3), 175 - 83
Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster; Kerver JW et al.; Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations . These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN) . The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages . In adults the increase in tolerance was not accompanied by an increase in overall ADH activity . However, there were changes in the distribution of ADH over the body parts . Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN . Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations . After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol . The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.

Prog Brain Res, 1992, 92, 25 - 37
Relationships among the FMRFamide-like peptides; Greenberg MJ et al.; The nuclear family of FaRPs (comprising those peptides that are, on compelling evidence, homologous) appears to be restricted to the protostome invertebrate phyla: i.e . Mollusca, Arthropoda, Annelida and Nematoda . Neither the origin nor the range of the family has been definitively established . That is, no genuine homologs have been demonstrated yet in the flatworms (though not for lack of trying), and neither the pseudocoelomate phyla related to the nematodes, nor the coelomate relatives of the annelids have been examined . The extended family of FaRPs (including peptides with little consistent sequence similarity beyond a penultimate Arg and an amidated hydrophobic residue at the C-terminal) exists in all phyla . Such a superfamily was probably first proposed by Morris et al . (1982), whose sequencing of SCPB suggested to them a class of peptides, ".. . the key unit for biological activity being Phe-A-Arg-B-amide (where A and B are also hydrophobic amino acids)." The ubiquity of the convergent FaRPs could reflect a conserved family of complementary heptahelical receptors requiring the arginyl residue for binding (Price and Greenberg, 1989) . But another selective advantage would be the protection provided by a penultimate Arg against certain deamidating peptidases, found so far in yeast and mammals (Jackman et al., 1990).

Folia Microbiol (Praha), 1992, 37(6), 413 - 20
Effect of increasing methanol concentrations on physiology and cytology of Candida boidinii; Volfova O et al.; Concentration of methanol in the medium strongly affected not only the physiology but also the cytology of Candida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rate D 0.1/h and at low pH 3.0 . The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S0 3 g/L) disappeared on increasing the methanol concentration in the inflow medium . The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium . The activity of catalase was relatively stable . At methanol concentration S0 > KI (KI equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria . The cells formed pseudomycelium with a thick septum between the mother and daughter cells.

Arch Tierernahr, 1992, 42(1), 1 - 9
Studies of the digestibility of pigs fed dietary sucrose, fructose or glucose; Ly J; Digestion of sucrose, fructose and glucose was studied in pigs with reentrant cannulas in the distal ileum or intact pigs fed diets formulated with sucrose, fructose and glucose as the major energy source and torula yeast . Sucrose and glucose were completely digested in the small intestine (apparent digestibility, 98.3 and 98.3%, respectively but a fraction of the dietary fructose escaped to the large intestine (adjusted value, 11.7%) . There was no carbohydrate in rectal contents of the pigs . There was no carbohydrate influence on the pre-caecal or total digestibility of nitrogen.

J Enzyme Inhib, 1992, 6(4), 271 - 82
Exploring the hexokinase glucose binding site through correlation analysis and molecular modeling of glucosamine inhibitors; Coats EA et al.; A series of N-substituted glucosamines has been designed, synthesized, and tested as inhibitors of yeast hexokinase . All derivatives exhibited competitive inhibition kinetics with respect to glucose . Quantitative structure-activity relationships were derived from the resulting inhibition data . The most significant equation demonstrated the existence of highly specific steric effects for the seven meta-substituted benzoylglucosamines included in the relationship . Molecular modeling of potential complexes between the inhibitors and the hexokinase substrate binding site strongly suggests that the steric effects arise from potential contacts with two amino acid residues lying in the region occupied by the amide substituents.

Korean J Intern Med, 1992 Jan, 7(1), 9 - 12
Prevalence of antibodies to hepatitis C virus in patients with various types of liver diseases; Kim KH et al.; BACKGROUND: Hepatitis C virus (HCV) is known to be a major cause of non-A, non-B hepatitis (NANBH) and is thought to be an important causative agent of serious liver disease . Recently the role of HCV in the development of various liver disease is suggested . METHODS: Sera from 222 patients with various liver diseases had been kept frozen at -20 degrees C until the test . Anti-HCV was detected using the ABBOTT HCV EIA Test System (ABBOTT Co., America) following the manufacturer's instructions . The assay uses a recombinant HCV antigen (C 100-3) synthesized in yeast . RESULTS: HCV antibodies (anti-HCV) were detected in 35 (31.5%) of 111 HBsAg-negative patients . The prevalence rate of anti-HCV was 61.9% (13 out of 21 patients) in chronic hepatitis, 29.1% (14 out of 48) in liver cirrhosis, 26.3% (5 out of 19) in hepatocellular carcinoma and 13% (3 out of 23) in acute hepatitis was far less (3 out of 111 patients, 2 . 7%) than that of HBsAg-negative patients (p < 0.01) . In this group, anti-HCV was detected in 2 (5.1%) out of 39 liver cirrhosis, 1 (1.9%) out of 52 chronic hepatitis, among them 47 were biopsy-proven chronic active hepatitis, and none of 20 hepatocellular carcinoma . CONCLUSIONS: These data suggest that, in Korea, 1) coinfection of HCV and HBV is infrequent, 2) HCV might be an important cause of HBsAg-negative chronic hepatitis, 3) HCV is seemed to be a less likely important factor associated with liver cirrhosis or hepatocellular carcinoma in HBsAg-negative patients, but further prospective study with a large population is necessary.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1084 - 8
Differences in substrate specificities of monoamine oxidase A from human liver and placenta; Tan AK et al.; The substrate specificities of monoamine oxidase (MAO) A isolated from human placenta and of human liver expressed in yeast have been compared in homogeneous preparations with respect to Vmax and Km values for natural and synthetic substrates and Ki values for competitive inhibitors . MAO A from these two sources is known to differ in at least 5 amino acid residues . While the Km and Ki values were found to be nearly identical in the enzymes from these two sources, the Vmax differed significantly on bulky synthetic substrates.

Cell, 1991 Dec 20, 67(6), 1181 - 94
Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins; Galaktionov K et al.; Two previously unidentified human cdc25 genes have been isolated, cdc25A and cdc25B . Both genes rescue a cdc25ts mutant of fission yeast . Microinjection of anti-cdc25A antibodies into HeLa cells causes their arrest in mitosis . cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold, in the absence of cdc2, by stoichiometric addition of either cyclin B1 or B2 but not A or D1 . Association between cdc25A and cyclin B1/cdc2 was detected in the HeLa cells . These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase, but also activate the cdc25 tyrosine phosphatase, of which cdc2 is the physiological substrate . A region of amino acid similarity between cyclins and tyrosine PTPases has been detected . This region is absent in cdc25 phosphatases . The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B.

Eur J Biochem, 1991 Dec 18, 202(3), 1021 - 7
Primary structure of a barley-grain aspartic proteinase . A plant aspartic proteinase resembling mammalian cathepsin D; Runeberg-Roos P et al.; Two enzymatically active heterodimeric forms of an aspartic proteinase, a putative 32 kDa + 16 kDa precursor form and a putative 29 kDa + 11 kDa mature form, are present in resting barley grains (Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J . & Mikola, L., 1990, in the press) . The cDNA corresponding to this enzyme has been cloned and sequenced . The full-length 1863-bp cDNA sequence codes for an open reading frame of 508 amino acids . The open reading frame consists of a 66-amino acid preprosequence and a 442-amino acid mature protein . Comparison of the N-terminal amino acid sequences of the enzyme subunits with the sequence of the cDNA clone indicates that the heterodimeric enzyme is translated as a proenzyme which is processed into two subunits . The localisation of the experimentally determined N-terminal amino acid sequences of all four subunits (32 kDa + 16 kDa and 29 kDa + 11 kDa) in the same transcript, as well as the detection of only one 2.0-kb mRNA on Northern blots from resting seeds, clearly indicates that the larger (32 kDa + 16 kDa) enzyme is an intermediate precursor form of the smaller (29 kDa + 11 kDa) enzyme . The processing pattern of the barley enzyme, which is the first sequenced plant aspartic proteinase, differs from that of all other known aspartic proteinases . The barley enzyme is highly similar to mammalian and yeast aspartic proteinases, especially to human and porcine cathepsin D . This similarity is clearly dispersed over two regions, separated by a dissimilar, barley-specific region of 104 amino acids.

Cancer Res, 1991 Dec 15, 51(24), 6712 - 4
Cloning of ALL-1, the locus involved in leukemias with the t(4;11)(q21;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13) chromosome translocations; Cimino G et al.; Chromosomal region 11q23 participates in a number of reciprocal translocations with specific regions of chromosomes 4, 9, 19, and others . These translocations are associated with acute lymphocytic leukemia and acute myelomonocytic, monocytic, and myelogenous leukemia . From a yeast artificial chromosome containing human DNA derived from 11q23 we cloned a DNA fragment which can be used as a probe to detect rearrangements in leukemic cells from the majority of patients with the t(4;11), t(9;11), and t(11;19) translocations . The breakpoints cluster in a small DNA region of less than 5.8 kilobases.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11393 - 7
Prohormone processing in Xenopus oocytes: characterization of cleavage signals and cleavage enzymes; Korner J et al.; In this study, we characterize the sequences required for the cleavage of prohormones in Xenopus oocytes . We demonstrate that the yeast alpha-factor and the Aplysia egg-laying hormone (ELH) precursors are not cleaved in oocytes following simple pairs of basic residues, such as Lys-Arg, but that the ELH precursor is cleaved following the consensus sequence Arg-Xaa-(Lys/Arg)-Arg . This motif is conserved among precursors that are cleaved in virtually all mammalian cell types . Mutations that generate this sequence in the alpha-factor prohormone also result in efficient processing within oocytes . Cleavage at this consensus sequence may be due to the action of the Xenopus homologues of mammalian furin.

Science, 1991 Dec 13, 254(5038), 1659 - 62
Targeting of the master receptor MOM19 to mitochondria; Schneider H et al.; The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP . Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor . The gene encoding MOM19 has now been isolated . The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol . MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP.

Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10735 - 9
Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias; Ziemin-van der Poel S et al.; Recurring chromosomal translocations involving chromosome 11, band q23, have been observed in acute lymphoid leukemias and especially in acute myeloid leukemias . We recently showed that breakpoints in four 11q23 translocations, t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13.3), were contained within a yeast artificial chromosome clone bearing the CD3D and CD3G gene loci . We have identified within the CD3 yeast artificial chromosome a transcription unit that spans the breakpoint junctions of the 4;11, 9;11, and 11;19 translocations, and we describe two other, related transcripts that are upregulated in the RS4;11 cell line . We have named this gene MLL (myeloid/lymphoid, or mixed-lineage, leukemia.

Nucleic Acids Res, 1991 Dec 11, 19(23), 6511 - 7
Coordinate expression of ribosomal protein genes in Neurospora crassa and identification of conserved upstream sequences; Shi YG et al.; The relative levels of rRNAs and ribosomal proteins are coordinately regulated by growth rate and carbon nutrition in Neurospora crassa . However, little is known about the mechanisms involved . To investigate the transcriptional regulation of ribosomal protein genes in N . crassa, we cloned and sequenced a ribosomal protein gene (crp-3) . The inferred crp-3 protein sequence shares 89% and 83% homology at its N-terminus with the yeast rp51 and the human S17 ribosomal proteins respectively . The crp-3 gene contains two introns, neither of which are conserved in position with the RP51 or the S17 genes . The crp-3 gene is present in a single copy and was mapped by RFLP analysis to the right arm of linkage group IV, near the cot-1 locus . Sequence comparisons of the upstream regions of the three sequenced crp genes revealed several common features . These include a 'Taq box' (consensus: ARTTYGACTT) at -39, a CG repeat (consensus: CCCRCCRRR) at -65, and a major transcription initiation site embedded in a purine rich region flanked by an upstream pyrimidine rich sequence . Using four N.crassa ribosomal protein genes as probes, we demonstrated that the levels of the four ribosomal protein mRNAs were closely coordinated during a nutritional downshift from sucrose to quinic acid.

Nucleic Acids Res, 1991 Dec 11, 19(23), 6441 - 7
The Trypanosoma brucei DNA polymerase alpha core subunit gene is developmentally regulated and linked to a constitutively expressed open reading frame; Leegwater PA et al.; As an initial step towards the characterization of replicative DNA polymerases of trypanosomes, we have cloned, sequenced and examined the expression of the Trypanosoma (Trypanozoon) brucei brucei gene that encodes the DNA polymerase alpha catalytic core (pol alpha) . The protein sequence contains the six conserved regions that have been recognized previously in eukaryotic and viral replicative DNA polymerases . In addition, we have identified a seventh region which appears to be conserved primarily in alpha-type DNA polymerases . The T.brucei DNA pol alpha core N-terminus is 123 and 129 amino acids smaller than that of the human and yeast homologue, respectively . The gene is separated by 386 bp from an upstream open reading frame (ORF) of 442 codons . Stable transcripts of the upstream sequence are detected in both dividing and non-dividing forms, while pol alpha transcripts are detected principally in dividing forms . Allelic copies of the T.brucei pol alpha region exhibit restriction site polymorphisms; one such sequence polymorphism affects the amino acid sequence of the T.brucei DNA pol alpha core . The T.brucei pol alpha region cross-hybridizes weakly with that of T.(Nannomonas) congolense and T.(Duttonella) vivax.

Biochemistry, 1991 Dec 10, 30(49), 11585 - 95
Effects of surface amino acid replacements in cytochrome c peroxidase on complex formation with cytochrome c; Corin AF et al.; Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase . The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins . The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography . The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme . The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase . The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c . This empirical finding localizes a primary recognition region critical to the dynamic complex . Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.

J Biol Chem, 1991 Dec 5, 266(34), 23175 - 84
Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor . Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation; Thomas LJ et al.; The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells . We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis . Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation . Two mutants were chosen for this analysis . The first mutant is defective at the first step of GPI anchor biosynthesis, i.e . in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor . Thus, it cannot form any GPI precursors or GPI-like compounds . Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma . These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation . The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis . Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression . Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis . When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored . However, N-linked glycosylation was either partially or completely corrected in different transfectants . Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins . These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

J Biol Chem, 1991 Dec 5, 266(34), 23334 - 40
The Drosophila RBP-J kappa gene encodes the binding protein for the immunoglobulin J kappa recombination signal sequence; Furukawa T et al.; We previously isolated a cDNA encoding the 60-kDa murine protein (RBP-J kappa protein) that specifically binds to the immunoglobulin J kappa recombination signal sequence . The RBP-J kappa gene is highly conserved in a wide variety of organisms including man, Xenopus, Drosophila, and yeast . We have isolated and characterized the Drosophila homologue of the RBP-J kappa gene . The Drosophila RBP-J kappa gene was mapped to the polytene region 35BC of chromosome 2 . The nucleotide sequence of this gene indicates that it is not one of the known genes located in the 35 BC region . The nucleotide and amino acid sequences of the Drosophila and mouse RBP-J kappa genes are 60 and 75% homologous, respectively . The central 248-residue regions of RBP-J kappa proteins of the two species are 93% homologous and include the 40-residue integrase motif . The Drosophila RBP-J kappa protein expressed in COS cells bound to the J kappa recognition sequence with the same specificity as the murine counterpart . These results suggest that Drosophila may have a site-specific recombination system which utilizes the immunoglobulin recombination signal sequence . Implications for evolution of immunoglobulin gene rearrangement were also discussed.

J Mol Biol, 1991 Dec 5, 222(3), 553 - 65
Characterization of the memory gene dunce of Drosophila melanogaster; Qiu YH et al.; The dunce (dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility . The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases or base-pairs) . To characterize these RNAs and to understand their sequence heterogeneity, we isolated and analyzed 29 new and independent cDNA clones representing the dnc RNAs . Restriction mapping, hybridization analysis and sequence determination of these cDNA clones and the corresponding genomic exons resolved these into six different classes . Exons defined by the cDNA clones are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs . The RNAs are transcribed from at least three initiation sites: two of these were mapped by parallel S1-nuclease and primer extension experiments . In addition, some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence . Altogether, the results indicate that the size and sequence heterogeneity of dnc transcripts results from transcription initiation at multiple sites, alternative splicing, and processes which generate different 3' ends . The existence of multiple protein products is suggested by the alternative use of exons which code for portions of the open reading frame . The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons and internal differences arising from the optional inclusion of a 39 base-pair exon and from the alternative use of two 3' splice sites separated by six base-pairs . Expression of a cDNA clone in yeast containing a large portion of the open reading frame produced cAMP PDEase activity identical in properties to the Drosophila enzyme affected by the dnc mutation . The results suggest that the remarkable structural complexity of dnc may reflect an intricate control of the spatial and/or temporal expression of various isoforms of cAMP PDEase.

Biochim Biophys Acta, 1991 Dec 2, 1129(1), 100 - 4
The most conserved nuclear-encoded polypeptide of cytochrome c oxidase is the putative zinc-binding subunit: primary structure of subunit V from the slime mold Dictyostelium discoideum; Rizzuto R et al.; A full-length 515 base pairs cDNA for cytochrome c oxidase subunit V of D . discoideum was isolated from a lambda gt11 expression library . The encoded polypeptide, whose identity was confirmed by partial protein sequencing, is 119 amino acids long (Mr = 13,352) and does not contain a cleavable presequence . The protein, which is homologous to human subunit Vb and yeast subunit IV, exhibits the highest degree of sequence conservation found among nuclear-encoded subunits of cytochrome c oxidase from distantly related organisms . All the invariant residues are clustered in two regions of the C-terminus which include the putative amino acids involved in the coordination of the Zn ion tightly associated to eukaryotic oxidase.

Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10421 - 5
A steroid-inducible gene expression system for plant cells; Schena M et al.; Promoters that allow the selective induction of gene expression in vivo constitute an important methodology in eukaryotic organisms such as yeast and the fruit fly, but to date no such system has been described for higher plants . Given the fact that mammalian steroid receptors can function as hormone-dependent inducers of gene expression in heterologous systems, the feasibility of using mammalian steroid hormones as selective inducers of plant gene expression was investigated . Here it is shown that the glucocorticoid receptor expressed in plant cells is capable of activating a test gene linked to glucocorticoid response elements, providing the transfected plant cells are treated with glucocorticoid hormone . Nanomolar concentrations of glucocorticoids are sufficient to induce gene expression more than 150-fold, without causing detectable alterations in the physiology of the cultured plant cells . These findings indicate that glucocorticoid induction of steroid-responsive promoters should provide a general method for regulating gene expression in plant cells and imply that such a system might ultimately function in whole plants such as Arabidopsis thaliana.

Gastroenterology, 1991 Dec, 101(6), 1680 - 91
Differential inhibition of individual human liver cytochromes P-450 by cimetidine; Knodell RG et al.; Cimetidine binds to cytochrome P-450 and inhibits hepatic metabolism of various drugs in humans . However, cytochrome P-450 is a family of enzymes rather than a single protein, and effects of cimetidine on individual human liver cytochromes P-450 have not been previously characterized . Metabolism of selected substrates and cimetidine-binding assays have been performed using human liver microsomes, purified human liver cytochromes P-450, and cytochrome P-450 complementary DNA-expressed yeast proteins to probe interaction of cimetidine with these individual enzymes . Cimetidine (3.0 mmol/L) in incubations reduced bufuralol hydroxylase activity by 80% and strongly inhibited microsomal nifedipine oxidation (23% +/- 13% of control activity) . The same concentration of cimetidine produced intermediate inhibition of cytochrome enzymes responsible for ethoxyresorufin deethylation and aniline hydroxylation (77% +/- 6% and 68% +/- 17% of activity in control microsomal incubations, respectively), but little effect on tolbutamide hydroxylation was observed . Concordantly, the calculated binding constant for the binding of cimetidine to a purified cytochrome P-450 with high tolbutamide hydroxylase activity was 4.4 mmol/L, whereas the calculated binding concentration constant for a purified cytochrome P-450-metabolizing nifedipine was 0.7 mmol/L . These studies show a high variability in the effect of cimetidine on drug metabolism by individual human liver cytochromes P-450 . In vitro studies using human liver microsomes and genetically engineered human cytochromes P-450 can be very useful in exploring important clinical questions of hepatic drug metabolism.

Southeast Asian J Trop Med Public Health, 1991 Dec, 22(4), 577 - 80
Intradermal hepatitis B virus immunization: immunogenicity and reactogenicity; Sirinavin S et al.; Mass immunization of hepatitis B virus (HBV) vaccine in adults is frequently demanded . However the high cost of conventional immunization is an obstacle to the provision of this vaccine . We investigated the serological response and adverse reactions following administration of a low-dose (1 or 2 micrograms of yeast-derived HBV vaccine (HB-VAX II, Merck, Sharp and Dohme) intradermally in young adults . Each 1 ml dose of the vaccine contained 10 micrograms of HBsAg protein . The study population included 58 female volunteers, aged 20-33 years, who were serologically-negative for HBV . They were alternately allocated to 1 microgram or 2 micrograms intradermal dose given by 2 experienced nurses as one or two 0.1 ml injections . Doses were given at 0, 1, and 6 months . Anti-HBs concentration was tested by enzyme-immunoassay on their sera obtained at 1, 6, and 7 months after the first dose . Positive seroconversion (anti-HBs greater than 10 IU/1) at 7 months was found in 90% (95% CL 79%, 100%) of the 1 microgram group and 96% (95% CL 89%, 100%) of the 2 micrograms group . Local reaction, a transient pigmented macule with an underlying nodule, was found in most volunteers but did not bother them . Intradermal HBV immunization could be an alternative strategy for mass immunization in young adults.

Appl Biochem Biotechnol, 1991 Dec, 31(3), 267 - 72
Eicosapentaenoic acid (EPA) production by Mortierella alpina ATCC 32222; Bajpai P et al.; Mortierella alpina ATCC 32222 grew well at 11 degrees C, as well as at 25 degrees C in a liquid medium containing glucose or linseed oil and yeast extract . High Eicosapentaenoic acid (EPA) yield was obtained at 11 degrees C . M . alpina cells did not produce EPA at 25 degrees C in the absence of linseed oil, whereas at 11 degrees C, EPA accumulation was noted in the absence of linseed oil . When grown at 11 degrees C for 10 d in a medium containing 2% linseed oil as carbon source, the mycelium yielded 435 mg/L EPA (20 mg EPA/g dry mycelia) with 5.1% in lipid fraction . By gradually increasing the concentration of linseed oil to 4%, yield of biomass and EPA were increased to 43 g/L and 596 mg/L, respectively.

Arzneimittelforschung, 1991 Dec, 41(12), 1265 - 76
Pharmacology of the potent new non-steroidal anti-inflammatory agent aceclofenac; Grau M et al.; Aceclofenac (2-{(2,6-dichlorophenyl) amine}phenylacetoxyacetic acid; CAS 89796-99-6) is a new orally effective non-steroidal anti-inflammatory agent of the phenylacetic acid group which showed remarkable anti-inflammatory, analgesic, and antipyretic properties . Hence, aceclofenac possesses a potent inhibitory activity in several models of acute and chronic inflammation in rodents, and resembles indometacin and diclofenac in its pharmacodynamic profile, being superior to naproxen and phenylbutazone . In addition, aceclofenac was found to be highly active against sodium urate-induced synovitis in dogs and adjuvant-induced polyarthritis in rats, both prophylactically and therapeutically . The analgesic effect of aceclofenac on the pain elicited by chemical and mechanical stimuli was nearly equal to or slightly better than that of indometacin and diclofenac . Fever induced by brewer's yeast injection in rats was also markedly suppressed by aceclofenac . In contrast, the acute gastric ulcerogenic activity of aceclofenac was about 2, 4 and 7-fold lesser than that of naproxen, diclofenac, or indometacin, respectively . As a consequence of its high anti-inflammatory activity and lower potential for gastric damage aceclofenac exhibited the most favourable therapeutic ratio in comparison with indometacin, diclofenac, naproxen, and phenylbutazone . These data indicate that aceclofenac could be a potent anti-inflammatory and analgesic agent with a wide margin of safety in clinical practice.

Chem Pharm Bull (Tokyo), 1991 Dec, 39(12), 3350 - 2
Mitogenic activity of Tulipa gesneriana lectins on mouse and human lymphocytes; Oda Y et al.; Tulipa gesneriana lectin-erythrocyte (TGL-E) which agglutinates mouse erythrocytes showed a potent mitogenic activity on mouse spleen cells and human peripheral blood lymphocytes, however, TGL-E had only slight mitogenic activity on mouse thymus cells . Its subunit alpha with a molecular weight (MW) of about 26,000 showed a potent mitogenic activity as did that of native lectin, but subunit beta with a MW of about 14,000 showed no activity, indicating that the mitogenic activity of TGL-E originates from subunit alpha . TGL-E stimulated T cell enriched spleen cells which passed through a nylon column, but not spleen cells from a nude mouse or spleen cells treated with anti-Thy 1.2 antibody and complement . Thus, TGL-E stimulates only mouse T cells but not B cells . The other lectin in tulip bulbs, Tulipa gesneriana lectin-yeast showed no mitogenic activity on mouse spleen, thymus cells or human paripheral blood lymphocytes.

Zhongguo Zhong Yao Za Zhi, 1991 Dec, 16(12), 719 - 22, 761
{Biological characteristics of Cryptoporus volvatus (peck) Hubb., a medicinal fungus}; Hua Q et al.; Through ecological investigations and experimental studies, it has been shown that C . volvatus is a fungus growing on decaying matter; the carbon source of culture medium comes from glucose, honey, fructose, mannitol, etc; and its nitrogen source from peptone, yeast powder, etc . The best ratio of carbon to nitrogen (C/N) is 20-25: 1, optimum pH 5.5-6.5 and optimal temperature 25-30 degrees C.

Cancer Metastasis Rev, 1991 Dec, 10(4), 301 - 10
Neurofibromatosis type 1 (NF1) gene: implication in neuroectodermal differentiation and genesis of brain tumors; Nishi T et al.; The gene responsible for neurofibromatosis type 1 (NF1), a common autosomal dominantly inherited disease, has been isolated . A region of NF1 gene product has been demonstrated to share structural and functional similarities with the mammalian GTPase activating protein (GAP) and the yeast IRA proteins . Thus, the NF1 protein is thought to play a role in signal transduction by stimulating the conversion of the Ras protein from a GTP-bound active form to a GDP-bound inactive form . The increased risk of malignant tumors in neuroectodermal tissues of NF1 patients may be caused by disruption of growth and differentiation regulatory functions of the NF1 gene . A second type of the NF1-GAP related domain (NF1-GRD) transcript, which has an extra 21-amino-acid insert in the center of the previously reported first type transcript, has been described . This insert significantly changes the hydrophilicity and secondary structure of the central region of NF1-GRD, therefore, suggesting it also changes its function . Alternative splicing is the most likely mechanism by which these two types of transcripts arise . The NF1-GRD alternative splicing has been shown to be intimately involved in differentiation of neuroectodermal tissues . Aberrant regulation of the alternative splicing may contribute to tumor formation in neuroectodermal tissue.

Genomics, 1991 Dec, 11(4), 991 - 6
Genetic and physical mapping around the properdin P gene; Coleman MP et al.; A CA repeat has been found on the human X chromosome within 16 kb of the gene encoding properdin P factor (PFC) and has been shown to be a highly informative marker . Two more polymorphic CA repeats were found in a cosmid containing DXS228 . The CA repeats, and other markers from proximal Xp, were mapped genetically in CEPH families and the likely order of markers was established as Xpter-(DXS7, MAO-A, DXS228)-(PFC, DXS426)-(TIMP, OATL1)-DXS255-Xcen . This places PFC in the region Xp11.3-Xp11.23, thus refining previous in situ hybridization data . Two yeast artificial chromosomes (YACs) (440 and 390 kb) contain both PFC and DXS426, and one of them (440 kb) also contains TIMP . This confirms the genetic order TIMP-(PFC, DXS426) . PFC and TIMP are located on the same 100-kb SalI/PvuI fragment of the 440-kb YAC . Given the genetic orientation of TIMP and (PFC, DXS426), this YAC can now serve as a starting point for directional walking toward disease genes located in Xp11.3-Xp11.2 such as retinitis pigmentosa (RP2) and Wiskott-Aldrich syndrome.

Genomics, 1991 Dec, 11(4), 1014 - 24
Mammalian hexokinase 1: evolutionary conservation and structure to function analysis; Griffin LD et al.; We have amplified and sequenced the complete coding region of bovine hexokinase isoenzyme 1 (HK1) from brain RNA with PCR primers selected for sequence conservation . The sequence information was analyzed to evaluate the evolutionary and structure-function relationships among the mammalian and yeast HK isoenzymes . Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HK1 outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of HK1 essential to enzyme function . The ATP-binding site in the catalytic half of the HK1 protein resembles nucleotide-binding regions from protein kinases, with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the loss of HK1 catalytic function in the regulatory domain . Sequence comparisons suggest that the 50-kDa mammalian and yeast glucokinases arose separately in evolution . In addition to providing valuable phylogenetic and structure-function insights, this work provides an efficient strategy for rapid cloning and sequencing of the coding regions for other HKs and related proteins.

Microbiol Rev, 1991 Dec, 55(4), 543 - 60
Organelle biogenesis and intracellular lipid transport in eukaryotes; Voelker DR; The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis . Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently . Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids . Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific . ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria . ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane . The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved . Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior . The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface . The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane . The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.

Chromosoma, 1991 Dec, 101(4), 199 - 205
Across the nuclear pores with the help of nucleoporins; Carmo-Fonseca M et al.; Proteins targeted to specific intracellular organelles such as mitochondria or the endoplasmic reticulum are able to cross membranes . Yet, to enter or exit the nucleus, proteins and RNA must pass through nonmembranous "gates" of the nuclear envelope, the nuclear pore complexes . Recently, the primary amino acid sequence of a few nuclear pore proteins (the nucleoporins) became available . Nucleoporins from mammals, amphibians and yeast are structurally homologous indicating that nuclear pore structures are evolutionarily conserved in the eukaryotic cell . The role of nucleoporins in nucleocytoplasmic transport is still unclear: are nucleoporins involved in decoding nuclear targeting signals or are they mere transporters? Although definite answers are not yet available, data are rapidly accumulating from several laboratories using a variety of approaches.

Nucleic Acids Res, 1991 Dec, 19(25), 7243 - 50
Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo; Blangy A et al.; We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA . Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element . When injected in greater concentration, an early arrest of embryonic development resulted . In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert . Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres . Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents . Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box . Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development.

Pediatr Infect Dis J, 1991 Dec, 10(12), 928 - 32
Pseudooutbreak of Candida guilliermondii fungemia in a neonatal intensive care unit; Yagupsky P et al.; During a 3-week period multiple blood cultures obtained from 14 Neonatal Intensive Care Unit infants and 3 Newborn Unit babies grew Candida guilliermondii, a yeast rarely associated with infections in humans . At the time of detection of positive cultures, most infants had been hospitalized for days or weeks for serious perinatal conditions and treated with antibiotics and intravenous hyperalimentation . Two critically ill premature infants from whom the yeast was isolated were given amphotericin B . In 7 other infants, however, yeasts were recovered on the day of birth, raising the question of pseudofungemia . Exhaustive interrogation on the blood culture practices revealed that when drawing blood for a culture from small infants, "butterfly" needles were often flushed with a diluted heparin solution to prevent blood clotting . Culture of a single lot of diluted heparin vials, prepared at the hospital pharmacy and distributed to the Neonatal Intensive Care Unit and Newborn Unit shortly before the onset of the epidemic, grew between 10,000 and 15,000 colony-forming units of Candida guilliermondii/ml . Removal of contaminated heparin vials and discontinuation of heparinization of needles used for blood cultures resulted in cessation of the epidemic . The present outbreak illustrates the difficulties in recognizing pseudoinfections in sick premature infants and the importance of intensive investigation and intervention during such an outbreak.

Am J Trop Med Hyg, 1991 Dec, 45(6), 695 - 701
Safety and immunogenicity of a recombinant sporozoite malaria vaccine against Plasmodium vivax; Herrington DA et al.; A recombinant Plasmodium vivax circumsporozoite (CS) antigen representing approximately 70% of the CS protein was expressed in yeast and adsorbed onto aluminum hydroxide for use as a malaria vaccine . In a study of safety and immunogenicity, 30 volunteers were divided into four groups of 5, 5, 10, and 10 individuals, and inoculated intramuscularly with 50, 100, 200, or 400 micrograms of vaccine, respectively . Primary vaccinations were followed by two booster immunizations at six weeks and six months . Overall, the vaccine was well tolerated . Following the third vaccination, one volunteer developed acute hepatitis of uncertain etiology that resolved without sequelae . All volunteers in the 400-micrograms group, and six of 10 in the 200-micrograms group generated IgG against P . vivax CS protein, as determined by Western blot using recombinant CS protein . However, the magnitude of the antibody response measured by indirect immunofluorescence of intact sporozoites or enzyme-linked immunosorbent assay against the recombinant protein was low, and responses could not be boosted . Antigen-driven replication studies using peripheral blood lymphocytes failed to detect proliferative responses specific to peptide sequences represented in the recombinant vaccine, except in one volunteer . Minimal humoral and cell-mediated immune responses developed in most recipients who received this recombinant CS vaccine.

Biochem J, 1991 Dec 1, 280 ( Pt 2), 475 - 81
Methanol oxidation and assimilation in Hansenula polymorpha . An analysis by 13C n.m.r . in vivo; Jones JG et al.; The metabolism of methanol was monitored in whole cells of the methylotrophic yeast Hansenula polymorpha by using {13C}methanol and n.m.r . in vivo . The main products observed under normal conditions were trehalose and glycerol, whereas cells that were starved before exposure to {13C}methanol also accumulated glutamate, glutamine and alanine; formate was also more prominent in spectra from starved cells . Cells exposed to high methanol concentration together with high oxygenation oxidized methanol extensively, leading to formaldehyde accumulation; label was not found in any subsequent metabolic products, indicating possible cell inactivation . {13C}Formate was incorporated into metabolic products in glucose-grown cells exposed to 150 mM-methanol for 3 h, but not in cells starved for 3 h, in which it was oxidized . At 21 degrees C such 3 h-starved cells showed a slower metabolism of {13C}methanol compared with those at 37 degrees C, and also converted methanol into formate rather than into assimilation products . The labelling pattern in trehalose from starved cells at 37 degrees C was consistent with methanol assimilation via the pentose phosphate pathway . Lack of appearance of labelled formaldehyde and formate during metabolism under normal conditions suggests that the linear oxidation pathway is not a major contributor to methanol oxidation; their appearance in extreme conditions suggests instead a more likely role in detoxification.

Am J Hum Genet, 1991 Dec, 49(6), 1361 - 71
Adrenoleukodystrophy: a complex chromosomal rearrangement in the Xq28 red/green-color-pigment gene region indicates two possible gene localizations; Feil R et al.; We have characterized a complex chromosomal rearrangement in band Xq28, in an adrenoleukodystrophy patient who also has blue-cone monochromacy . A 130-kb region upstream from the color-vision pigment genes was isolated as yeast artificial chromosome or cosmid clones . Another Xq28 sequence, not included in the above region, was obtained by cloning a deletion breakpoint from the patient . Using probes derived from the cloned sequences, we have shown that the rearrangement affects the color-pigment genes and includes two deletions, most likely separated by a large (greater than 110-kb) inversion . One deletion encompasses part of the pigment gene cluster and 33 kb of upstream sequences and accounts for the patient's blue-cone monochromacy . If this rearrangement also caused ALD, the disease gene would be expected to lie within or close to one of the deletions . However, deletions were not detected in a 50-kb region upstream of the red-color-pigment gene in 81 other ALD patients . Two CpG islands were mapped, at 46 and 115 kb upstream from the pigment genes.

Curr Genet, 1991 Dec, 20(6), 495 - 502
The group IIB intron from the green alga Scenedesmus obliquus mitochondrion: molecular characterization of the in vitro splicing products; Winkler M et al.; In the presence of high molar salt concentrations, the mitochondrial group IIB intron (rI1) from the green alga Scenedesmus obliquus is capable of splicing in vitro . After establishing the optimal conditions for RNA processing the in vitro splicing products were unequivocally identified in self-splicing experiments by Northern hybridization analysis employing 3'end-labelled RNAs or exon- and/or intron-specific probes . Finally, two trans-esterification products were identified by sequencing of the spliced RNA . From our data we conclude that the processing of group II introns from both algal and yeast mitochondria is preceded by identical consecutive trans-esterification steps . The predicted secondary and tertiary structure of intron rI1 of S . obliquus contains all the motifs necessary for optimal self-splicing and which are characteristic of other group IIB introns from different species.

Genomics, 1991 Dec, 11(4), 799 - 805
Genome mapping with anchored clones: theoretical aspects; Ewens WJ et al.; As part of our effort to construct a physical map of the genome of Arabidopsis thaliana we have made a mathematical analysis of our experimental approach of anchoring yeast artificial chromosome clones with genetically mapped RFLPs and RAPDs . The details of this analysis are presented and their implications for mapping the Arabidopsis genome are discussed.

Acta Crystallogr B, 1991 Dec 1, 47 ( Pt 6), 1020 - 2
Characterization of two crystal forms of cytochrome c from Valida membranaefaciens; Day J et al.; Two different crystal forms of cytochrome c from the yeast Valida membranaefaciens have been grown by vapor diffusion from polyethylene glycol 4000 . Both have been characterized by X-ray diffraction . The first crystal form was grown at 277 K but proved unstable and dissolved when the temperature was increased above 288 K . This crystal was of triclinic space group P1 and had unit-cell dimensions of a = 68.4, b = 35.7, c = 33.24 A, alpha = 116.9, beta = 114.9, gamma = 75.0 degrees . Following dissolution of the triclinic crystals, the second crystal form also grew at 277 K but remained stable even at room temperature . It was of orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 27.95, b = 63.68, and c = 64.28 A . Although rather small in size, both crystal forms diffracted X-rays well and the resolution of the patterns extended beyond 3 A.

Arch Biochem Biophys, 1991 Dec, 291(2), 363 - 70
DCCD binds to cytochrome b6 of a cytochrome bf complex isolated from spinach chloroplasts and inhibits proton translocation; Wang YD et al.; Radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD) was bound selectively in a time- and concentration-dependent manner to cytochrome b6 of an enzymatically active cytochrome bf complex isolated from spinach chloroplasts . Maximum labeling of cytochrome b6 was observed with 30 nmol DCCD per nmol cytochrome b6 in the cytochrome bf complex incubated for 30-60 min at 12 degrees C . After incubation of the cytochrome bf complex with DCCD under these conditions, the rate of proton ejection in the complex reconstituted into liposomes was decreased approximately 65-70% when compared to controls; however, under these same conditions the rate of electron transfer through either the soluble bf complex or the complex reconstituted into liposomes was only decreased around 20% . These results suggest that the mechanism of proton translocation through the cytochrome bf complex of spinach chloroplasts is similar to that of the cytochrome bc1 complex from yeast mitochondria in which proton pumping but not electron transfer is also inhibited by DCCD (D . S . Beattie and A . Villalobo, 1982, J . Biol . Chem . 257, 14,745-14,752).

J Biol Chem, 1991 Nov 5, 266(31), 20913 - 21
The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts; Prat-Gay G et al.; High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system . High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+ . At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM) . The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged . The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation . On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol . At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure . The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition . Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle . The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.

Gene, 1991 Nov 15, 107(2), 329 - 33
Cloning and sequencing a cDNA encoding human ribosomal protein S25; Li ML et al.; A full-length cDNA clone has been isolated from a cDNA library prepared from mRNA of adriamycin-resistant human leukemia HL60 cells . The nucleotide sequence of this cDNA has been determined and the protein coded for by the gene identified . The cDNA encodes a polypeptide of 125 amino acids (aa) with a deduced Mr of 13750 . The deduced aa sequence of this protein has 56% homology to yeast ribosomal protein S31 . Western-blot analysis using antibodies directed against a synthetic peptide based on the deduced aa sequence identifies the gene product as the human ribosomal protein S25.

Gene, 1991 Nov 15, 107(2), 205 - 12
Characterization of the structure and transcription of an ubiquitin fusion gene from maize; Chen KQ et al.; We have identified a maize ubiquitin (Ubi) fusion gene (UBF9) by screening a maize W22 genomic phage lambda library with a short (16-nucleotide) oligodeoxyribonucleotide probe derived from the sequence for the extension sequence of a yeast UB13 fusion gene . UBF9 consists of an UB monomer sequence (228 bp long) joined to an extension sequence (237 bp long) . The extension sequence encodes a protein of 79 amino acids which shares extensive identity with similar extension aa sequences found in yeast, humans, barley and Arabidopsis thaliana . UBF9 encodes a small-size class of Ubi mRNAs in the maize tissues investigated . The UBF9 transcript is present in high levels in maize endosperm tissues 22 days after pollination . Genomic Southern blots of maize inbred W22 DNA indicate that the fusion gene sequences are present in multiple copies in the maize genome . Primer extension experiments indicate that the transcription start point is located at 80 bp upstream from the translation start codon of UBF9 . Two 37-bp tandem repeated A + T-rich sequences are found in the 5'-flanking region of UBF9 . The A + T-rich sequences share the motif, AATATTTTATT, which is present in a diverse set of plant genes.

Biochim Biophys Acta, 1991 Nov 14, 1115(1), 6 - 14
Dictyostelium discoideum acidic ribosomal phosphoproteins: identification and in vitro phosphorylation; Prieto J et al.; Four acidic phosphoproteins from the ribosomes of the slime mold Dictyostelium discoideum have been identified and partially characterized . These proteins are selectively released from ribosomal particles by salt/ethanol washes, have low molecular weight and acidic pI, and tend to aggregate in solution to form homodimers . These features correspond to proteins of different origins that have been included in the conserved family of eukaryotic A-ribosomal proteins, and, therefore, we have named them Dictyostelium ribosomal proteins A1, A2, A3 and A4 . We also demonstrate that Dictyostelium ribosomal A-proteins are specifically phosphorylated in vitro by a type II casein kinase previously identified in Dictyostelium . Isoelectric focusing separation has permitted us to identify four proteins (or P-proteins) that may consist of the phosphorylated forms of A-proteins . A-proteins from Dictyostelium and yeast do not present immunological cross-reactivity . Dictyostelium A-proteins contain, therefore, some specific features in their amino acid sequence that distinguish them from other members of the conserved eukaryotic A-protein family; this conclusion is coherent with data deduced from the nucleotide sequence of cDNA clones encoding two Dictyostelium A-proteins (P1 and P2) which we have recently reported.

Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1453 - 9
Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes; Lithgow T et al.; Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes . When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of {alpha-32P}GTP, a 52 kDa protein was radiolabelled, whereas {alpha-32P}ATP did not label this protein . GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes . Fractionation of mitochondrial membrane vesicles into outer membranes, inner membranes and contact sites between outer and inner membranes showed that the GTP-binding activity was highly enriched in contact sites, the location at which preprotein import is believed to occur . A protein of almost identical size was also found to be labelled in mitochondria from yeast.

Nature, 1991 Nov 7, 354(6348), 80 - 2
Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1; Bischoff FR et al.; The product of the gene RCC1 (regulator of chromosome condensation) in a BHK cell line is involved in the control of mitotic events . Homologous genes have been found in Xenopus, Drosophila and yeast . A human genomic DNA fragment and complementary DNA that complement a temperature-sensitive mutation of RCC1 in BHK21 cells encode a protein of relative molecular mass 45,000 (Mr 45K) which is located in the nucleus and binds to chromatin . We have recently isolated a protein from HeLa cells that strongly binds an anti-RCC1 antibody and has the same molecular mass, DNA-binding properties, and amino-acid sequence as the 205 residues already identified . HeLa cell RCC1 is complexed to a protein of Mr 25K . We have shown that this 25K protein has a sequence homologous to the translated reading frame of TC4, a cDNA found by screening a human teratocarcinoma cDNA library with oligonucleotides coding for a ras consensus sequence, and that the protein binds GDP and GTP . We have referred to this protein as the Ran protein (ras-related nuclear protein) . In addition to the fraction of Ran protein complexed to RCC1, a 25-fold molar excess of the protein over RCC1 was found in the nucleoplasm of HeLa cells . Here we show that RCC1 specifically catalyses the exchange of guanine nucleotides on the Ran protein but not on the protein c-Ha-ras p21 (p21ras).

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9628 - 32
Direct selection: a method for the isolation of cDNAs encoded by large genomic regions; Lovett M et al.; We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions . The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone . Nonspecific hybrids are eliminated and selected cDNAs are eluted . These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles . This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene . Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment . More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched . We confirmed that this cDNA was encoded by the yeast artificial chromosome . Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library . DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins . Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene . The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9623 - 7
cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments; Parimoo S et al.; Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies . We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR . The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments . By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9493 - 7
Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement; Yao MC et al.; A method for transforming Tetrahymena has been established earlier, but its application has been limited because of the lack of selectable markers other than the rRNA-encoding DNA (rDNA) . Mutations in the yeast ribosomal protein L29 gene (CYH2) are known that confer cycloheximide resistance . We have cloned and sequenced the homologue of this gene from both a wild-type and a cycloheximide-resistant (ChxA) strain of Tetrahymena . Surprisingly, a comparison shows that the ChxA mutation is not present in the CYH2 homologue . We therefore created the yeast mutations in the Tetrahymena gene by site-directed mutagenesis and used them to transform Tetrahymena either with or without linking to an rDNA vector . All clones transformed by the rDNA vector also became resistant to cycloheximide when the rDNA contained the engineered mutant genes . Without the rDNA vector, the mutant genes transform approximately 1% of injected cells to become resistant to cycloheximide . DNA analysis indicates that transformation occurs by replacement of the host sequence and not by random integration of the injected sequence . The replacement occurs to some but not all copies of this gene in the polyploid macronuclear genome . Thus, transformation in Tetrahymena occurs by specific sequence replacement, and the injected mutant genes can serve as dominant selectable transformation markers in this organism.

J Nutr, 1991 Nov, 121(11 Suppl), S1 - 7
Contribution of the dog to the science of nutrition; Carpenter KJ; In the 19th century when the basic principles of nutrition were established, the main work was done first in France and then in Germany . In each country dogs were the overwhelming choice as the model experimental animal . In France the complexity of nutritional requirements first came to be appreciated and the inadequacy of gelatin as a substitute for muscle protein was identified . In Germany quantitative balance procedures for nutrients were developed and it was shown that balance could be achieved at many levels after a period of adaptation . In the U.S.A . at the beginning of this century, Russell Chittenden showed that dogs could do well when fed low protein diets so long as they contained some nonprotein factors that were provided by meat and milk . On the basis of that work Joseph Goldberger developed a diet which produced a condition analogous to pellagra in dogs . This led to the discovery that yeast was a potent preventive of the disease and to the eventual identification of niacin as the primary active factor . Work in Britain with dogs as models for experimental rickets gave apparently conflicting results, with either environmental or dietary changes apparently protecting from the disease . Further work showed that calciferol could be obtained either by irradiation of the skin or by the consumption of another animal's store . Lastly, Edward Mellanby's continued work on the rachitic effect of cereals led to the spin-off finding that wheat flour improved with nitrogen chloride, although nontoxic to rats, was responsible for the problem of canine hysteria in dogs that had developed in the 1930s and 1940s . Its use by millers was than banned.

J Exp Med, 1991 Nov 1, 174(5), 1203 - 8
Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals; Barr PJ et al.; Pfs25 is a sexual stage antigen of Plasmodium falciparum that is expressed on the surface of zygote and ookinete forms of the parasite . Monoclonal antibodies directed against native Pfs25 can block completely the development of P . falciparum oocysts in the midgut of the mosquito vector . Thus, this 25-kD protein is a potential vaccine candidate for eliciting transmission-blocking immunity in inhabitants of malaria endemic regions . We have synthesized, by secretion from yeast, a polypeptide analogue of Pfs25 that reacts with conformation-dependent monoclonal antibodies, and elicits transmission-blocking antibodies when used to immunize mice and monkeys in conjunction with a muramyl tripeptide adjuvant . Our results suggest the further evaluation of recombinant DNA-derived Pfs25 in transmission-blocking vaccination studies in humans.

Eur J Biochem, 1991 Nov 1, 201(3), 615 - 25
Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase . Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme; Adolph HW et al.; 1 . The steady-state parameters kcat and Km and the rate constants of hydride transfer for the substrates isopropanol/acetone; (S)-2-butanol, (R)-2-butanol/2-butanone; (S)-2-pentanol, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(II)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(II)LADH . 2 . A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E in equilibrium E.NAD in equilibrium E.NAD.R1R2 CHOH in equilibrium E.NADH.R1R2CO in equilibrium E.NADH in equilibrium E . 3 . On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions . 2-Butanone and 2-pentanone reductions revealed (S)-2-butanol (86%) and (S)-2-pentanol (95%) as the major products . 4 . The observed enantioselectivity implies the existence of two productive ternary complexes; E.NADH.(pro-S) 2-butanone and E.NADH.(pro-R) 2-butanone . All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined . These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent kcat/Km values for the two different ternary-complex configurations of 2-butanone . Additionally, these data have been used for computer simulations of the corresponding reaction cycles . Calculated, simulated and experimental data were found to be in good agreement . Thus, the system (S)-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADH-catalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism . 5 . The effects of Co(II) substitution on the different steps of the kinetic pathway have been investigated . The free energy of activation is higher for alcohol oxidation and lower for ketone reduction when catalyzed by Co(II)LADH in comparison to Zn(II)LADH . However, the free energies of binding are affected by metal substitution in such a way that the enantioselectivity of ketone reduction is not significantly changed by the substitution of Co(II) for Zn(II) . 6 . Evaluation of the data shows that substrate specificity and stereoselectivity result from combination of the free energies of binding and activation, with differences in binding energies as the dominating factors . In this regard, the interactions of substrate molecules with the protein moiety are dominant over the interactions with the catalytic metal ion.

Carcinogenesis, 1991 Nov, 12(11), 2175 - 9
Effect of deficiencies of selenium and vitamin E alone or in combination on the induction of mammary carcinogenesis by 1-methyl-1-nitrosourea; Thompson HJ; Experiments were conducted to determine the dietary levels of selenium and vitamin E that could be fed chronically to induce a deficiency of one or both nutrients . It was observed that diets containing less than 0.02 mg Se/kg and 3 IU vitamin E/kg induced a deficiency without drastically altering growth performance or survival . Once established, these dietary conditions were used to investigate the effect of single or combined deficiencies of selenium and vitamin E on the promotion phase of mammary carcinogenesis induced by 1-methyl-1-nitrosourea (MNU) . In the carcinogenesis experiment, 21 day old female Sprague-Dawley rats were fed a torula yeast formulated diet deficient in selenium and vitamin E until they were 50 days of age . At that time, each rat was injected i.p . with 12.5 mg MNU/kg body weight . Seven days thereafter, rats were randomly assigned to one of four treatment groups that were deficient or adequate in selenium and/or vitamin E . The experiment was terminated 30 weeks after carcinogen treatment . In comparison to rats fed the adequate diet, final cancer incidence and number were higher and cancer latency shortened in rats consuming the diet deficient in both selenium and vitamin E . Single deficiencies of either nutrient failed to significantly alter the tumorigenic response.

Mol Cell Biol, 1991 Nov, 11(11), 5701 - 9
Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans; Dowzer CE et al.; The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented . The gene contains no introns . The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein . The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein . A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition . Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation . This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.

J Cell Biol, 1991 Nov, 115(3), 795 - 808
ras isoprenylation is required for ras-induced but not for NGF-induced neuronal differentiation of PC12 cells; Qiu MS et al.; We have used compactin, an inhibitor of mevalonate biosynthesis, to block p21ras posttranslational modification and membrane association in PC12 cells . Previous studies have demonstrated a requirement for isoprenylation for mitogenic effects of activated p21ras in mammalian cells and for function of RAS gene products in yeast . Immunoprecipitation of {35S}methionine-labeled p21ras from PC12 cell homogenates confirmed that the processed p21ras species is missing from compactin-treated PC12 cells . Immunoprecipitation from particulate and cytosolic fractions of PC12 cells confirmed that compactin blocks p21ras membrane association: p21ras is confined to the cytosol fraction . Induction of neuronal differentiation and ornithine decarboxylase (ODCase) transcription by oncogenic p21N-ras does not occur in compactin-treated cells indicating that activity of oncogenic p21N-ras expressed in PC12 cells is abolished by compactin treatment . Thus, p21ras isoprenylation or association with the membrane appears to be required for early responses and neuronal differentiation attributable to p21ras activation . In contrast, blockade of p21ras isoprenylation and membrane association by compactin treatment did not significantly reduce PC12 cell responses to NGF . Responses examined included rapid phosphorylation of tyrosine hydroxylase, rapid induction of ODCase expression, survival in serum-free medium and neuronal differentiation . Compactin blocked growth factor-induced rapid changes in cell surface morphology but did so whether this response was induced by NGF or by EGF . These results indicate that functional p21ras is not necessary for responses to NGF which in turn implies that if a ras-dependent NGF signal transduction pathway exists, as has been previously suggested, at least one additional ras-independent pathway must also be present.

PCR Methods Appl, 1991 Nov, 1(2), 111 - 9
Capture PCR: efficient amplification of DNA fragments adjacent to a known sequence in human and YAC DNA; Lagerstrom M et al.; We have devised a procedure, termed capture PCR (CPCR), that permits the rapid isolation of DNA segments situated adjacent to a characterized nucleotide sequence . In this procedure, a DNA sample is restriction-digested and a linker, comprising two base-paired oligonucleotides, is added to the ends by ligation . Multiple extension reactions are performed using a biotinylated primer derived from the known sequence, permitting the subsequent isolation of extension products on a streptavidin-coated support . The enriched fragments are amplified exponentially using another specific oligonucleotide, hybridizing 3' to the biotinylated primer in combination with one of the linker oligonucleotides, now functioning as a PCR primer . The convenience of CPCR is greatly enhanced by using a novel streptavidin-coated manifold, which is constructed so that it projects into each individual well of a microtiter plate . The procedure permits the simultaneous isolation of fragments from large numbers of DNA samples and minimizes the risk of contamination between reactions . We have applied this method to identify DNA sequences located downstream of known sequences in the human genome . The technique has also been used to identify end fragments of sequences cloned in a yeast artificial chromosome (YAC) vector . The reactions can be initiated directly from yeast colonies and provide access to DNA sequence information for these end fragments in a minimal number of steps . With the aid of the present technique, we have isolated over 100 end fragments from YACs derived from the human X chromosome . Isolated end sequences have been used to order YAC clones into a contig.

Int J Dermatol, 1991 Nov, 30(11), 795 - 8
Epidemiology of chronic paronychia in a skin hospital in Singapore; Chow E et al.; A retrospective epidemiologic study of 110 patients with chronic paronychia (CP) showed a female-male ratio of 2.3:1, whereas the ratio of patients attending the same clinic was 1.1:1 (p less than 0.001) . The peak age range of patients with CP (40-49 years) generally was greater than that of the general dermatologic patients (20-29 years) . Seventy-seven percent of the patients with CP were "manual workers," of which 48% were homemakers . Chronic paronychia was more common on the right fingers than the left fingers . The most commonly affected fingers were the right thumb (62%), followed by the right middle finger (52%), left thumb (57.6%), and left middle finger (51.5%) . Mechanical trauma appears to be an important predisposing factor in CP . Sixty-two percent of 68 patients who had nail fold smears had positive findings for budding yeast cells, suggestive of candidal infection . All of the six patients for whom nail fold bacterial cultures were performed had positive results for enteric flora.

Am J Trop Med Hyg, 1991 Nov, 45(5), 533 - 8
Phase I clinical trial of a recombinant malaria vaccine consisting of the circumsporozoite repeat region of Plasmodium falciparum coupled to hepatitis B surface antigen; Vreden SG et al.; R16HBsAg is an experimental recombinant malaria vaccine consisting of 16 repeats of a four amino acid sequence (Asn-Ala-Asn-Pro or NANP) of the circumsporozoite (CS) protein of Plasmodium falciparum expressed as a fusion protein with the recombinant hepatitis B virus surface antigen (HBsAg) produced by yeast cells . Twenty male volunteers were experimentally vaccinated with the product, as well as with two doses of the commercial recombinant HBsAg vaccine Engerix B (Smith Kline Beecham Biologicals, Rixensart, Belgium) at intervals during a period of 18 months . No serious side effects were observed . Circulating antibodies to recombinant CS antigen (R32tet32) developed in all volunteers and persisted in most cases over ten months . Anti-HBs antibody production was poor initially, but a single dose of the commercial hepatitis B vaccine was sufficient to elevate these titers to high levels in all but two volunteers.

Biokhimiia, 1991 Nov, 56(11), 1960 - 8
{Regulation of Ca(2+)-dsRNA for proliferation and terminal differentiation processes of human fibroblasts and HeLa cells}; Zakharian RA; Ca2+ complexes of dsRNA, poly(dA) and poly(dT) of yeast low molecular weight RNA produce a pronounced mitogenic effect on human fibroblasts at early stages of fibroblast proliferation in culture . At later stages of cell cultivation Ca(2+)-dsRNA stimulates terminal differentiation by inducing the synthesis of proteins characteristic of the postmitotic population of human fibroblasts undergoing terminal differentiation . Ca(2+)-dsRNA produces a stimulating effect on c-fos and c-jun gene transcription in fibroblasts and HeLa-S-3.

Zentralbl Veterinarmed B, 1991 Nov, 38(9), 681 - 4
Assessment of lymphocyte and phagocytic functions in goats treated with glucan; Benda V et al.; The effect of glucan, a biological response modifier of yeast origin, on different immune functions was studied after the intramuscular application in goats . The simultaneous administration of glucan with human serum albumin or tetanus toxoid significantly stimulated the antibody production to both antigens differing in their thymus dependency . Similarly, the phagocytizing activity of the blood polymorphonuclear leukocytes measured by the reductase colorimetric assay significantly increased one week after the glucan treatment . However, suppression of T-lymphocyte function in experimental animals was determined by the lymphocyte transformation test particularly in response to phytohemagglutinin and concanavalin A . The results of the study indicate that glucan can modulate some elements of the ruminant immune response.

Appl Environ Microbiol, 1991 Nov, 57(11), 3310 - 6
Metabolism of phenanthrene by Phanerochaete chrysosporium; Sutherland JB et al.; The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80 . After cultures were grown with {9-14C}phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting . Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside . Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra . Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P . chrysosporium.

Mycopathologia, 1991 Nov, 116(2), 105 - 12
Diagnostic potential of IgA coated Candida cells in mucous membrane candidiasis; Polonelli L et al.; The significance of in vivo IgA coated yeast cells for the diagnosis of candidiasis of the oral and vaginal mucosal membranes was evaluated by direct immunofluorescence in 70 patients with or without clinical symptoms, shown to be positive for yeast growth in the cultural test . Most of the patients with clinically suspected candidiasis of the mucosal membranes gave positive results by serologic assays in contrast to the majority of symptomless patients . The diagnostic approach proved to be essentially consistent with the clinical signs, persistence of infection, response to antifungal therapy and quantitative cultural data.

Mol Endocrinol, 1991 Nov, 5(11), 1571 - 7
Estrogen receptor variants in clinical breast cancer; McGuire WL et al.; We have used the screening techniques of chemical mismatch cleavage, single stranded conformational polymorphism, and gel retardation to discover a number of estrogen receptor RNA variants in clinical breast cancer tissues . We have found basepair insertions, transitions, and deletions as well as alternative splicing, yielding deletions of exon 3, 5, or 7 . Using a yeast transactivation assay we have discovered receptors with outlaw function, including both a dominant-positive receptor, which is transcriptionally active in the absence of estrogen, and a dominant-negative receptor, which is itself transcriptionally inactive, but prevents the action of normal estrogen receptor . These variants could have clinical significance, helping to explain breast tumor behavior and patient outcome.

J Assoc Off Anal Chem, 1991 Nov-Dec, 74(6), 957 - 60
Monitoring aldehyde production during frying by reversed-phase liquid chromatography; Lane RH et al.; Acrolein (2-propenal) and other low molecular weight aldehydes (LMWAs) formed by degradation of the frying medium (triglycerides) were monitored by liquid chromatography (LC) during preparation of fried items . LMWA contents of coatings from codfish and of doughnuts and their volatiles that codistill with steam are monitored by trapping the vapors and distillate from the food matrix in a 2,4-dinitrophenylhydrazine solution . The resulting hydrazones are partitioned from the aqueous phase, first into isooctane and then into acetonitrile for LC analysis . The hydrazones are separated and quantified on a C18 reversed-phase column with acetonitrile-water as the mobile phase . LMWAs are confirmed by gas chromatography/mass spectrometry . No difference was found in LMWA content in coatings from fish fillets fried at 182 or 204 degrees C . Cake doughnuts were higher in acrolein content than yeast-raised doughnuts prepared under similar conditions . Freshness of the frying medium, frying time, and batch size did not seem to influence LMWA production from doughnuts . Results indicated that most of the LMWAs formed codistilled with steam during frying rather than remaining with the food item.

Cancer Genet Cytogenet, 1991 Nov, 57(1), 109 - 19
DNA sequences of chromosome 21-specific YAC detect the t(8;21) breakpoint of acute myelogenous leukemia; Kearney L et al.; The t(8;21)(q22;q22) is a nonrandom translocation specifically marking blasts of acute myelogenous leukemia (AML) with undifferentiated phenotype . The breakpoint on chromosome 21 involved by this rearrangement has been precisely localized relative to cloned DNA markers by physical and genetic linkage analysis enabling the use of positional cloning for its isolation . Yeast artificial chromosome (YAC) clones for loci proximal (D21S65) and distal (ERG) to the (21q22) breakpoint have been developed and their chromosome 21 origin and location relative to the breakpoint has been established . By using in situ hybridization analysis, a 240 kb YAC clone for the D21S65 locus clearly identified both derivative chromosomes of the (8;21) translocation in metaphase spreads of leukemia blasts with the rearrangement . The characterization of the DNA sequences contained in this 240 kb YAC can reveal the functional consequences of their derangement in leukemia with abnormalities of the (21q22) region.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9794 - 8
Reverse transcriptase encoded by a retrotransposon from the trypanosomatid Crithidia fasciculata; Gabriel A et al.; The long interspersed nuclear element (LINE)-like elements are a distinct family of eukaryotic transposons that contain a long open reading frame with limited sequence homology to retroviral reverse transcriptases . Unlike many retrotransposons, they lack long terminal repeats . The mechanism by which LINE-like elements move within the genomes of their hosts remains speculative . We have used an unusual approach to express and detect enzymatic activities associated with Crithidia retrotransposable element 1 (CRE1), a site-specific LINE-like element found in the insect trypanosomatid Crithidia fasciculata . A chimeric gene fusing the yeast retrotransposon Ty1 and the CRE1 open reading frame is constructed and then overexpressed in yeast . Fusion proteins are packaged into virus-like particles, which can be partially purified and directly analyzed for enzymatic activity . Here we demonstrate that CRE1 encodes an RNA-directed DNA polymerase . These data provide direct biochemical evidence that this widely distributed class of retrotransposons encodes reverse transcriptase and sets the stage for a detailed understanding of the mechanisms involved in LINE-like element transposition.

Mutat Res, 1991 Nov, 255(3), 227 - 40
The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks; Knox RJ et al.; The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents . This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent . A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR) . The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents . There were no significant differences in the binding of these agents to the DNA of WS or WR cells . All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks . WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells . Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink . Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic . Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants . The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells . In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells . The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair.

Genomics, 1991 Nov, 11(3), 720 - 9
Isolation, localization, and physical mapping of a highly polymorphic locus on human chromosome 11q13; Eubanks JH et al.; A highly polymorphic repetitive sequence, D11S533, was isolated by oligonucleotide hybridization from an arrayed chromosome 11q-specific cosmid library . The DNA sequence of this element was determined and found to consist of a repetitive degenerate hexanucleotide sequence {T(Pu)T(Pu)T(Pu)}n extending over 438 bp . Southern blot analysis demonstrated that this element is relatively unique in the human genome . This sequence can be detected by amplification using the polymerase chain reaction (PCR) with oligonucleotide primers complementary to unique sequences flanking the repetitive element . This sequence displays a high degree of polymorphism, and analysis of 15 individuals demonstrated at least 10 alleles ranging in size from 300 to 900 bp . Fluorescence in situ hybridization was used to localize this sequence to 11q13 (FLpter 0.60 +/- 0.02) . Pulsed-field gel electrophoresis and the isolation of yeast artificial chromosomes established the long-range physical map surrounding the locus . Because various alleles of this polymorphic sequence can be easily detected by PCR amplification, this probe has potential usefulness in genetic linkage mapping as well as identity testing.

J Leukoc Biol, 1991 Nov, 50(5), 423 - 6
Suppression of human macrophage function in vitro by delta 9-tetrahydrocannabinol; Specter S et al.; The ability of macrophages to function in the presence of delta 9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, was evaluated . THC added to macrophage cultures prepared from human peripheral blood inhibited macrophage spreading and phagocytosis of yeast . The effects of THC were concentration dependent, with inhibitory effects observed from 10 to 1 micrograms/ml or lower . These results suggest that macrophages are more sensitive to THC than are lymphocytes because macrophage functions were inhibited by THC at concentrations that did not affect lymphocyte function . Thus, inhibition of lymphocyte function(s) by THC could be attributed to a direct effect of the drug on macrophages which indirectly results in lowered lymphoid cell activity.

Hum Genet, 1991 Nov, 88(1), 112 - 4
Regional mapping of 22 microclones around the adenomatous polyposis coli (APC) locus on chromosome 5q; Hampton GM et al.; A previously described genomic library constructed from microdissected DNA has been used to generate a large number of probes around the adenomatous polyposis coli (APC) gene at 5q22 . A total of 202 clones were hybridised directly onto a somatic cell hybrid panel containing two APC-related interstitial deletions . Of 75 microclones that gave clear hybridisation signals, 22 independent clones mapped into the region common to both deletions . In addition, 4/22 of the markers are conserved in rodent DNA . These clones should provide a valuable resource for screening cDNA libraries and cloning the DNA around the APC gene in yeast artificial chromosomes.

Mol Pharmacol, 1991 Nov, 40(5), 699 - 706
Inhibition of eukaryotic DNA topoisomerase I and II activities by indoloquinolinedione derivatives; Riou JF et al.; With the aim of obtaining new inhibitors of topoisomerases, we have evaluated various heterocyclic quinone derivatives for their ability to induce topoisomerase I (Topo I)- or Topo II-associated DNA breaks, using P388 cell nuclear extract . Several compounds belonging to the indolo{3,2-c}quinoline-1,4-dione series have been shown to possess DNA-cleavage activity . Further analysis using purified Topo I and II preparations has indicated that the members of the series stimulate cleavable complex formation of both Topo I and II . 3-Methoxy-11H-pyrido{3',4':4,5}pyrrolo{3,2-c} quinoline-1,4-dione (AzalQD), one of the most active members of the series, stimulates cleavable complex formation and inhibits the catalytic activities of both eukaryotic Topo I and II, with, however, less potency than camptothecin and etoposide . Topo I cleavage site patterns for AzalQD and camptothecin were found to be nearly identical, with, however, some differences in cleavage site intensities . Use of filter binding assays also indicates that AzalQD is at least 10 times more potent against Topo I than against Topo II . Structure-activity relationships of indoloquinolinedione derivatives have been established and have shown that Topo I and II inhibitions are strongly linked, with a dose-selective preference towards Topo I . AzalQD does not display detectable DNA-unwinding properties . AzalQD induces a preferential cytotoxicity for the yeast strain JN2-134 bearing the human top1 gene under the control fo the GAL1 promoter, indicating that Topo I inhibition is responsible for the yeast cytotoxicity . These data indicate that AzalQD and its structural analogs represent a new distinct class of eukaryotic Topo I and II inhibitors.

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